Gas chromatographic and mass spectrometric studies of synthetic and naturally occurring ceramides

Chem. Phys. Lipids 5 (1970) 44-79 © North-Holland Publ. Co., Amsterdam.

GAS C H R O M A T O G R A P H I C AND MASS S P E C T R O M E T R I C S T U D I E S OF S Y N T H E T I C AND N A T U R A L L Y O C C U R R I N G C E R A M I D E S KARIN SAMUELSSON and BENGT SAMUELSSON Department of Neurology, Karolinska Sjukhuset and Department of Medical Chemistry, Royal Veterinary College, Stockholm, Sweden Sphingolipids have been studied extensively with respect to the structures of the long chain bases (LCB)1-7) and of the fatty acids 7-11) obtained on hydrolysis. However, the structures of the individual molecular species of ceramides resulting from combination of the various fatty acids and LCB have not been determined earlier. This review summarizes recent work in our laboratory on the use of gas chromatography-mass spectrometry for separation and identification of synthetic ceramides. Furthermore, studies on naturally occurring ceramides, either free or as constituents of sphingomyelins, are described.

Gas-liquid chromatography of ceramides Ceramides were prepared by direct coupling with sphingosine or sphinganine using carbodiimide to activate the carboxylic acid. The ceramides were converted to TMS derivatives with hexamethyldisilazane and trimethylchlorosilane in pyridine 12). An F & M 400 gas chromatograph equipped with a hydrogen flame ionization detector was used. The column (a 1.8 m × 3 m m i.d. U-shaped silanized glass column) was packed with 2% OV-1 on 100-120 mesh Gas Chrom Q (both from Applied Science Laboratories Inc., State College, Pa.). The column (conditioned at 350 °C for 24 hr) had an efficiency of 2 500-3000 theoretical plates. In order to avoid leakage and bleeding at the high temperatures employed, the column was inserted with new O-rings and septum and set at the working temperature in the afternoon before the next day's experiment. The resolution obtained under different conditions was expressed as the minimum carbon number difference between two ceramide derivatives, which could be separated with base line resolution in the region LCB 18:1-20:0 to LCB 18:1-22:0 (AC values) 13) (table 1). With a column o f 2 % OV-I on gas 44

45

SYNTHETIC AND NATURALLY OCCURRING CERAM1DES TABLE 1

Effect of carrier gas and temperature on resolution Gas flow rate

Carrier gas

Resolution, AC~cB 18:1-20:0-2z:o

(ml/min)

320 °C

310 °C

135 90 75 135

0.9 0.7 0.6

0.7 0.6

Helium Helium Helium Nitrogen

1.0

c h r o m Q o p e r a t e d at 320 °C the m a x i m a l resolution achieved c o r r e s p o n d e d to a AC value o f 0.6. C e r a m i d e s containing m o n o u n s a t u r a t e d fatty acids were t b u n d to give the same resolution as their s a t u r a t e d analogues. The use o f nitrogen instead o f helium c o n s i d e r a b l y decreased the resolution. Similar effects have been observed with triglycerides 14). The response o f the h y d r o g e n flame i o n i z a t i o n detector was expressed as R D R (relative d e t e c t o r response per weight unit). It was f o u n d t h a t R D R for the c e r a m i d e derivatives (even c a r b o n n u m b e r fatty acids 16:0 to 24:0) was a l m o s t inversely p r o p o r t i o n a l to the n u m b e r o f c a r b o n a t o m s o f the constituent fatty acid (fig. 1). Previous studies have shown t h a t R D R for

bd 03 Z

SPHINGOSINE

°a

1.&'

~

1.2'

CERAMIDES

e,,-

ml/min N; 135; 310°

2 1.0. W

~ 0.6.

He; 135; "*'----* He; 135; ~ H e ; 90; ~ H e ; 90; -'He; 75;

o

tu 0.6"

o.,~-

320 ° 310° 320 ° 310 °

320*

,,_1 Ill

0.2"

16":o le:O 2o:0 2~:o 2~:o FATTY

ACID

Fig. 1. Effect of carrier gas type and flow rate and column temperature on the relative detector response (RDR) of the di-O-trimethylsilyl ether derivatives of ceramides containing sphingosine and the fatty acids 16:0, 18:0, 20:0, 22:0 and 24:0.

46

K. S A M U E L S S O N A N D B. S A M U E L S S O N

methyl esters of fatty acids is practically constant for long-chain acids la, 16). However the R D R for short-chain fatty acids decreased considerably with decreasing chain length due to the increasing percentage of carbonyl content. A constant R D R was also obtained for several trimethylsilyl ether derivatives of steroids differing with respect to the nature of substituents17). For triglycerides, however, it has been reported that R D R decreases for compounds larger than trimyristin14). This finding was interpreted to be due to mist formation caused by low volatility. In the present investigation it was found that increased carrier gas flow rate (tested from 75 to 135 ml/min), with temperature constant, resulted in increased RDR. With the carrier gas flow rate constant, the R D R was higher at 320°C than at 310°C. Furthermore a compound eluted with the same retention time at 310 °C and 320°C, obtained by adjusting the carrier gas flow rate, had a significantly higher R D R at the lower temperature. These results indicated that a higher temperature could increase R D R by decreasing the retention time but that this effect was counteracted by increased thermal destruction. On the basis of the characteristics of the flame ionization detector found for other compounds [see e.g.15-17)] and the results obtained in the present investigation it seems likely that the lower R D R for ceramides with longer fatty acids is due to thermal destruction. The detector response for all of the ceramides studied (even carbon number

1.8001 /

Z O U.I nr r',-"

o

(j, LLI I-UJ O ILl .,<

P_, c,-

1600"° LCB18:1-16:0 / ~/ 18:1-18:0 //~/* 1+400 •~LCB LCB18:1-20:0 / / / / " °LCB18:l-22:0 / //P ' 2°° .,c+ ,000 /2/J [

800

/

Y

/

~

600 400 f 200 011 0~I2 013 01~ 01i~ MICROGRAMS INJECTED

Fig. 2. Linearity of detector response for the di-O-trimethylsilyl ether derivatives of ceramides containing sphingosine and the fatty acids 16:0, 18:0, 20:0, 22:0 and 24:0. Operating conditions as listed in fig. 1.

SYNTHETIC AND NATURALLY OCCURRING CERAMIDES

47

fatty acids 16:0 to 24:0) was found to be linear within the range investigated (0.05/~g to 0.5/~g) (fig. 2). This indicates that the decrease in the detector response for the longer chain ceramides was approximately proportional to the amount of sample injected. Ceramides containing monounsaturated fatty acids gave practically identical R D R as the corresponding saturated analogues. The precision obtained was acceptable down to 0.05/zg/injection. The retention time of the ceramides was related to that of triglycerides and expressed as triglyceride carbon units (TGCU). There was a linear relationship between the number of carbon atoms of the ceramides and the T G C U values (fig. 3). The nonselective phase used (OV-1) gave slightly lower T G C U

LLJ O'J Z O O_

LCB 18:1-16:0

IdJ i,'v" it,.

2u LI,I U,,I O

LCB 18:1-18:0 LCB 18:1-20:0

LCB 18:1-22:0

'~

;

;

LCB 18:1-2ZD:0

1'o

1'2

MINUTES

Fig. 3. Gas-liquidchromatography of di-O-trimethylsilylether derivativesof sphingosine ceramides containing the fatty acids 16:0, 18:0, 20:0, 22:0 and 24:0. Column: 2 ~ OV-1, column temperature: 320°C. Carrier gas He. Gas flow rate 90 ml/min.

values for sphingosine ceramides containing unsaturated fatty acids as compared with saturated fatty acids and this was also found for sphingosine ceramides as compared with sphinganine ceramides. However the differences in retention time were too small to allow complete separation on the basis of degree of unsaturation. Similar qualitative data were obtained for ceramides containing 2-hydroxy acids (table 2).

48

K. SAMUELSSON AND B. SAMUELSSON

TABLE2 Triglyceride carbon units for the di-O-trimethylsilyl ether derivatives of sphingosine and sphinganine ceramides Triglyceride carbon units Sphingosine

Sphinganine

Fatty a c i d

Normal

2-hydroxy

Normal

2-hydroxy

16:0 18:0 18:1 19:0 20"0 22: 0 22:1 23:0 24:0 24:1 25:0

37.4±0.1" 39.5 ±0.1 39.2±0.2 40.5 ± 0.1 41.6 ± 0.1 43.6 ± 0.1 43.3 ±0.1 44.6 ±0.1 45.7 ± 0.1 45.5 ± 0.2 46.7 ±0.1

38.1 ±0.1 40.0 ± 0.1

38.0 ±0.1 40.0 ±0.1

42.0 ± 0.1 43 9 ± 0.1

37.7±0.1 39.7 ±0.2 39.1 ±0.3 40.7 ± 0.1 41.7 j 0.1 43.7 ± 0.1

45.8 ± 0.2

44.7 ±0.1 45.8 ± 0.2

41.9 ± 0.2 43.9 :L-0.1 45.8 ± 0.2

46.7±0.1

* S.D., five determinations

Gas chromatography-mass spectrometry of ceramides

Normalfatty acid ceramides The LKB gas chromatograph-mass spectrometer, model 9000, equipped with an 1.2 m glass column (3 mm i.d.) with a packing of 1 ~ OV-I (nonpolar silicone phase) on 60-80 mesh Gas Chrom Q was used. The column was conditioned at 350°C for 24 hr. The column temperature was 275°C and the flash heater and separator temperatures were about 300 °C 18). The mass spectrum of the TMS derivative of N-stearoyl sphingosine will first be considered in detail (figs. 4 and 5). The molecular weight is indicated by ions at m/e 694 (M - 15), m/e 619 (M - 90), and m/e 606 (M - 103) formed by elimination of a methyl group, trimethylsilanol, and the terminal - - C H z - - O - - S i ( C H 3 ) 3 , respectively. Eliminations involving the fatty acid residue produce ions at m/e 426 (M +(b + 1)) and m/e 336 ( M - ( b +1 +90)) by loss of stearoyl am±de and, in the latter case, also trimethylsilanol. Loss of stearoyl am±de plus CH 3(CH2)12 ( M - (b + 1 + e)) gives an ion at m/e 243; cleavage between C-2 and C-3 with charge retention on the main sphingosine fragment results in an ion at m/e 311. The same cleavage but with charge retention on the other part of the molecule gives rise to an ion appearing at m/e 398 ( M - a ) . Another fragmentation involving loss of the main part of the sphingosine molecule produces an ion at m/e 471 ( M - ( a - 7 3 ) ) , which

SYNTHETIC

AND NATURALLY

OCCURRING

CERAMIDES

(1

o\

R

5:

33NVaNfWl

R 311v13kl

, 0

P

49

50

K. SAMUELSSONAND B. SAMUELSSON

is tentatively ascribed to cleavage between C-2 and C-3 and transfer of the TMS group to the remaining fragment. An ion appearing at m/e 247 is also tentatively interpreted to be formed by this reaction and, in addition, //cleavage of the stearoyl residue with transfer of hydrogen from the 7-carbon atom, i.e. (M - ( a - 73 + 224)); the additional elimination of trimethylsilanol gives rise to an ion at m/e 157.

i

c

,i4

C~ b

e

~ H3(CH2)I2

CH=CH or --CH 2--CH 2a

Fig. 5.

I

CH~CH

dC H 2 - - O

TMS

Some important fragmentsfor structure determinationof ceramides by mass spectrometry.

In order to obtain experimental evidence for the fragmentations proposed above, we prepared the corresponding derivative of N-perdeuterostearoyl sphingosine and subjected it to GLC-mass spectrometry. The mass spectrum is shown in fig. 6. The fragments (M-15), (M-90), and (M-103) now appeared at m/e values 35 units higher than those of the corresponding nondeuterated derivative. This is also seen for other fragments that retain the fatty acid residue, i.e. ( M - a ) and ( M - ( a - 7 3 ) ) . However, fragments formed by elimination of the fatty acid, ( M - (b + 1)), ( M - ( b +1 +90)), and (M - ( b + 1 +e)) have the same m/e values as in the nondeuterated derivative. Evidence for the//-cleavage of the stearoyl residue with transfer of hydrogen from the 7-carbon atom to the carbonyl oxygen in the formation of fragments ( M - ( a - 73 + 224)) and ( M - ( a - 73 + 224 + 90)) was also obtained. The reaction involves retention of three hydrogens originating in the fatty acid; it was accordingly found that the fragments mentioned above appear at m/e 250 and 160 instead of 247 and 157, respectively. It is evident from the results described above that the molecular weight of a ceramide can be determined from the fragments M - 15, M - 9 0 and M - 103, and the nature of the LCB from the fragments formed by elimination of the acylamide, with or without trJmethylsilanol, and by cleavage between C-2 and C-3. In the example discussed above these fragments appear at role 426, 366, and 311, respectively. The structure of the fatty acid moiety can be deduced from the fragments formed by cleavage between C-2 and C-3

SYNTHETIC AND NATURALLY OCCURRING CERAMIDES

E

I u')

(0

C.)

I

.

v

7r

"r

0

rO-O (.,) ll I (.,)

r~

.9

I-

u t~ I

I uo

q) I o"i

i

I,,,3-

GD

O

v

8

O

+O

o o

6 3DNVOnBV

¢M

3MIV73~I

51

52

K. SAMUELSSON

i

AND

i

B. SAMUELSSON

i

~ ¢ _ . . . oo

,--

~

.o

u

v-

o tJ

l

,

T

"1-

I I I..~ - - Z

0 --tO

=

~

¢0--0

~ u

.N

a

~J,

~

v ~

z

~

~-.T

"~®

E

-~.o°

,

~/

g

~~

~

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o

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6

3^11V-13~I

53

SYNTHETIC A N D NATURALLY O C C U R R I N G CERAMIDES

with or without transfer ofa TMS group. The resulting ions appear at role 471 ( M - (a-73)) and m/e 398 ( M - a), respectively. Some of the fragmentations described above have also been observed by Gaver and Sweeley, who characterized N-acetyl derivatives of sphingosine and sphinganine 19). The mass spectrum of the TMS derivative of N-stearoyl sphinganine is shown in fig. 7. The molecular weight is again obtained from the eliminations M - 1 5 , M - 9 0 , and M - 1 0 3 , which give rise to ions appearing at m/e 696, 621 and 608, respectively. Information on the structure of the LCB is provided by M - d (cleavage between C-2 and C-3) appearing at m/e 313, whereas charge retention on the other part of the molecule gives a fragment (m/e 398) which can be used to determine the structure of the fatty acid residue. Other "fatty acid fragments" are formed by additional elimination of a trimethylsilyloxy group ( M - ( a + 8 9 ) ) or capture of the TMS group ( M - ( a - 7 3 ) ) and appear at m/e 309 and 471, respectively. An ion that is absent from the mass spectrum of the sphingosine ceramide derivatives but that is relatively prominent in the sphinganine series appears at m/e 217. This ion most probably consists of C-1 to C-3 of the LCB from which acyl amide has been eliminated.

$PH[NGAN]NE FATTY

ACID

C26:0

100% {M-(b*t*c) 217

IM-(a. 69))

IM-d}

C20:0

465 3i3 423~28/ t~82

2/*7

J2?2//260

6~2,/705

7801

313 337

i2;=1=;o

C18:0

527

&37

365 395 /*28

2721/,/20o

217 2/.7

0% loo%

I

555

I./~,o, I

217 2/.7

0% %00%

Mw = 739

[,/

313

C22:0 Mw = 767

(M-103) 720 (M-90) (M-15) 733 808

313 217

0% 100%

583

(M-a)

,/] Y I S ~1°

0% 100%

C2/.,:0 Mw= 795

(M-(a-?3))

/421 451 (M-(b,I) V ~2S ~,93 /,8~=

313

Mw = 828

45/*

i

/*09

367

I

66~ 677

[/

752

i

~99

1.26

] I,,-\, L..,2,

6~6,7"649 72/* ,

t

313

3~

Mw = 711

2t'/ 2/*7

339 381 398

0% 100%

C16:0 Mw = 683

/*Tt l

313

217 2/*7 281311 353 0% 12321260" 1 3 2 6 ~ 3701

20O

Fig. 8.

CERAMIDES

300

443 /.28[

400

Mass spectrometric data for T M S

500

6611 5•0,/S93 I 600 700

800 m/e

derivatives o f sphinganine ccramides.

54

K. SAMUELSSON AND B. SAMUELSSON

In order to provide data for the identification of ceramides of natural origin and to facilitate the interpretation of fragmentation processes, two series of ceramides have been prepared, one with sphingosine as base and the other with sphinganine as base. In both cases the fatty acid residues are saturated normal acids ranging from C16 to C26. The results have been summarized in figs. 8 and 9 and table 3, where the fragments that are important for structure determination of ceramides are summarized. TABLE 3

Some important fragments for structure determination of ceramides by mass spectrometry "Molecular weight fragments"

"LCB fragments"

M-15

M--(b÷l)

M -- 90

M -- (b

M--103

M

"Fatty a c i d fragments"

Sphinganine derivatives

M-a

Sphingosine derivatives

M--(b-] 1+c) M - - ( b + l + e )

89) M--(a--73)

-F- ] -F- 9 0 )

M -- (a --

d

For explanation of portions a-d of the molecule, see upper right-hand corner of fig. 4; e is CHa(CH2h2 from sphinganine.

SPHINGO5INE FATTY

ACID

(M-ib,H) L26

(M-d) 31} C26:0

(M-(b.l.el)

M W = 821

I (M-{b.l.90))

Z~3

CERAMIDE5 [M-a)

510

]

(M-{a-?3}) 503 (M'1031~ (M-90) [

I00% MW = 793

(M-15)

~731

006

4,2

311

C24:0

71,

,6

415 I L,L,0

336 C22:0

s~5

.~

,,o3

,,.

426

MW=765

527

662 675

L

336

,., /

?50

1~25

C20:0 Mw = 737

3tl 364 I00%

722

426

MW = 709

47~

606

\ ,/619

L

I

I

2~7

1336

2~3 ~'-.c. 258 [~.e/ 326 300

"

?

'

370

311

20o F i g . 9.

63~ ,~6~?

311

C18:0

C16:0 MW = 681

49]9

396

[

426

J

1,3 1

4()0

5,8

--

" (~

500

s91

; "

'

600

"6 6 6

i

700

800

role

Mass spectrometric data for T M S derivatives of spLdngosine ccramides

SYNTHETIC AND NATURALLY OCCURRING CERAMIDES

55

It is evident from fig. 8 that in the sphinganine derivatives the "LCB fragments" M - (b + 1) and M - d appear at the same m/e value in all members of the series, whereas the "fatty acid fragments" M - a , M - ( a - 7 3 ) , and M - ( a + 89) are shifted 28 units between neighboring members of the series. This is also true for fragments appearing at m/e values 451,468, and 493 in the C26 homologue and the molecular weight fragments M - 15, M - 9 0 , and M - 103. Three ions that appear at m/e 217 ( M - ( b + 1 +c)), 232, and 260 are the same for all of the homologues of the sphinganine series. The sphingosine derivatives (fig. 9) show similar groups of fragments. Thus the "LCB fragments" M - ( b + 1), M - ( b + 1 + 90), and M - d appear at the same m/e value in all of the homologues. The "fatty acid fragments" M - a and M - ( a - 73) and "the molecular weight" fragments appear at m/e values that differ by 28 units between homologues. This is also seen for the fragments appearing at m/e 451,468, and 493 in the C26 homologue, and since the same ions are present in the sphingosine and sphinganine series they can probably be referred to as "fatty acid fragments" although no structures have as yet been assigned. Two ions appearing at m/e 243 and 258 in all of the sphingosine homologues are absent from the sphinganine series and might be important for the identification of the former series. An ion at m/e 247 is present in all of the homologues of both the sphingosine and sphinganine series.

Ceramides containing 2.hydroxy acids2°) The ceramides were synthesized by carbodiimide coupling of 2-acetoxyacids with D, L erythro-sphingosine or D, L erythro-sphinganine followed by mild alkaline hydrolysis z0). The trimethylsilyl ether derivatives were prepared as described above. Fig. 10 shows the mass spectrum of the 1,3,2'-tri-O-TMS derivative of N-(2'-hydroxystearoyl)-sphingosine. The designation of fragments is given in the structural formula of this figure. Many ions are analogous to those seen for ceramides with normal fatty acids. The molecular ion, m/e 797 is of low intensity and the molecular weight is more readily determined from the ions (M - 15), m/e 782; ( M - 90), m/e 707; (M - 103), m/e 694; ( M - 2 × 90), m/e 617 and ( M - 103 - 9 0 ) , m/e 604. These are presumably formed by elimination of " C H 3 from one of the TMS groups, a trimethylsilanol, the terminal C H 2 - - O - - S i ( C H 3 ) 3 radical, two molecules of trimethylsilanol and C H z - O--Si(CH3) 3 plus one molecule or trimethylsilanol, respectively. The spectrum has a prominent base peak at m/e 486. This can be visualized to be formed by homolytic cleavage of the C-C bond between C-2 and C-3 after initial charge localization on the nitrogen at C-2. Cleavage of the same bond with charge localization on the oxygen at C-3 is presumably involved in

~j Ix

--

z

a:

.~

b

i

~o

-,-

2x99)

~oo

zz=

~

t.g.

150

Fig.

200

]0.

Mass

250

.~i.L,

I/

~

I

(

~5o

I ~,:~355 ~

- -

a

"

b 2)

~

406

. ~ , , , t .... L .

~zy

M 1 _

(t) (M-(b.1.99))

/

3

429

300

~5o

r

,

450

.

500

., . . ... .

485 '

M-a) 504

-

. . .

i

613

6o0

2x

bi~(M-2xg~

(M-(a-82:~ 586

~

550

--

3 1--2

-

,

d

~o

.

sphingosine.

600

sphingosine

650

and

MW=797

M 99 (-

'

750

i

824.. 80C role

~.

MW=824

~o' ,~'

- -~, - 797

(M- 5)

40 . . . .

c.3q~CH3

~ -1-CH1 -CH2 --(CH2)¼CH3 I

, , i2 . . . . . . . . . .

L

700

~

6.94 ~

CH3~I CH3

CH3(CH2 12 ~-CH=CH--~H~--(~H--CH2OS~(CH3)3

a

, . . . .1 ~. . . l .~ .. . . . . . . .

~5o

1,3,2'-tri-O-trimethylsilyl-N-(2"-hydroxy-stearoyI)

400

500

(M-a- 9)

[~ 1 89%

559

-

,Te--15

. . . . . . . . . = ~ .I. 5 ,~ ~ ~. l _ t

f

1,3,2'-d27-tri-O-trimethylsilyl-lN-(2'-hydroxystearoyl)

of(a)

350

)

. L ~. ~ L I . A _ L

(M-(b,13 444

Jk

I

}

[M-a)

(b.h7:~ (M a t6) |

(M-(b.~ _ 7

. I , ~ _ I , J . L . L L, J .. . . . . .L... . h. ~. . . .d _ . k ~

295

320

spectrum (b)

(M-(b.1.e)J263

(

.~...,I,._[,L

M-(a-82 (g 1-99)

25o

_JLL_

i ~

(M-(b.1.e))245

~ _ ,. . L. . . .. . ... . .. . .I , ~.

-


. .., . ..... . .

100

25

75

100

,z-~u z

,,._J,L . . . . .~. . . . . . .

,oo

75

IO0

0 Z

0 Z > Z

g ~a

.z

SYNTHETIC AND NATURALLY

OCCURRING

57

CERAMIDES

CHa(CH2)12--CH=CH--CH-y----CH--CH2OSi(CHa)3

IJ

I

O

dq-NH

I -J

I

CH3SiCH 3

C--CH--(CHz),CHa

[

II I 0 I

CH3

0

Si(CH3)3 Fig. 11. Proposed formation of the ion (M -- (a -- 73)),

m/e 559.

the formation of the ion appearing at role 31 I. The ion appearing at m/e 599, ( M - ( a - 7 3 ) ) is probably also formed by cleavage between C-2 and C-3 but in this case the odd electron on the nitrogen is paired by transfer of a trimetbylsilyl radical as shown in fig. 11. Transfer of a hydrogen atom to the nitrogen in ( M - ( a - 7 3 ) ) followed by

SPHINGOSINE

FAT T¥ 26h:0

N-,~ 598 311 I (~-Cb+l~(f) (*,,1-a89) (ivl-(b +T-909~ 426\1 ,~3£ Co~2)509 ~q~÷73)...

(M-(b~1÷e~

wt=909

rnol

CERAMIDES

AC I D

2042z'3~ ~

N-e,-'73)) 671. (M-2xgO)?29

~-90) 819 8O6/

~-15~

894

24h:0 311

2~

mol wt =881

791

7"78./

B66

22h:0 243

mol wt=853

426

311

,

I

.

~,

t

4~

G,sl

i s~

~38

~o/673

20h:O

wt=B25 _.

tool

wt= 797 0

wt=769

mot .

.

.

.

.

.

.

I

.

.

|

o'/

722~5

a~

31t "

/

1

~32z45

i

~

4327

426

TOO'/]

16 h:O

632/645

587

/

1 8 h:O

mo{

.

2042z"~

~?0

559 I

617

707

6o4 /

694 /

782

]458 243

~

245

"<'~8

311 299,

34.,

4*6426 |

531

~3_~/~ ~.'%2

i

576589

~;"./

679

794

~ /

430

14h:0

100/°/j

......

o7 100

200

311

~,

~ . . . . . . 3O0

5013 948~ 5z.8/ zOO

500

651

638 / 600

726 700

Fig. 12. Mass spectrometric data for TMS derivatives of 2'-hydroxy sphingosine ceramides.

800

900 m/e

58

K. SAMUELSSON

AND

B. SAMUELSSON

cleavage of the bond between C-2 and the nitrogen could yield the ion at

m/e 444 (b +1 +73). An ion at m/e 426, ( M - ( b + 1)) is assumed to arise by elimination of acylamide. Additional elimination of one molecule of trimethylsilanol, m/e 336, ( M - ( b + 1)-90), is also seen regularly. The ion at m/e 372, ( b + 2 ) is presumably formed by transfer of two hydrogens to fragment (b). Cleavage between C-I' and C-2' with charge retention on the oxygen at C-2' is probably involved in the formation of m/e 327 (f), which seems to be specific for 2'-hydroxy acid ceramides. The ion ( M - ( b + 1 +e)) at m/e 243, resulting from elimination of acylamide and CH3(CH2)12, is present in sphingosine ceramides containing normal fatty acids or 2-hydroxy acids, but is absent in sphinganine ceramides. In order to obtain mass spectrometric data, and to aid the interpretation of fragmentations, two series of homologous ceramides containing 2-hydroxy acids ranging from 14h:0 to 26h:0 were prepared. The mass spectrometric data for sphingosine ceramides is shown in fig. 12 and for sphinganine ceramides in fig. 13. Two types of ions can be seen, namely ions that appear

SPHINGANINE

FATTY

CERAMIDES

ACID

26h:0

(M~Tl+c~)

moI wt=911

155 DO'J

24h:0

(M~'o+l)) (M {a73) (g !)) \ (f) I 335 348 419 \439

245

~ 313

i

4

8

(b 4~$'7)4

509 (M (g-'l) (b*l+T~%581 /, 8(M a) /~600

1

(M 90)

(M 103 9O) X/M 2x90)

~-~)31

808

(M) (M 15;/ 896 /

643 i

mol wt=883

780 793

703 453

866 8 8 ~

615

22h:0 mol wt=855

662 /675

752 1765

840 J8

20h:O tool wt=827

724 ~7

647

8~2 8

2

7

18h:O mol wt=799

696 313

784

369

16h:0 tool wt=771 27]

313

341

503

14h:O 5 25~

mol wt=743 100

200

Fig.

13.

I 43O 36 388 / , 348 428 ~32 3O0

400

563 50O

640 6O0

728 700

Mass spectrometric data for TMS derivatives of 2'-hydroxy sphinganine ceramides.

800

900 role

z

b

150

,38

129

173

I ,82

15"('~

200

250

Fig. 14.

300

~-d) 322

307

(f)

400

428

406

~-a-96)

450

, .... 500

.

!~ ~i~

(M-~-62) -99)

500

~

503

/ i',,,

/(.-,~

(M.e~2 / (M-a)

450

<~.,.7~

(M-(~-73} -9C~

.

53

,

550

.

-

650

(b) ],3,2'-d~7-tri-O-trimethy]sily]-N-(2'-hydroxystearoyl) sphinganin¢.

I

0

CH

700

• '

7~o ....

role

86o'g~'

(M-181 808 {M)

MW=826

800

M) It 799

b

MW=799

CH2--EH2)¼CH 3

750

CH3

CH3SliCH3

O

C

NH

(M 112) 714 [{M-99)

700

709 ~L [

CH3

CH3~CH 3

. . . . . . . . . . . . . . 600 650

[M-(g-0) 602 (M°112-99)

586 (M-(ar 82}

600

{M-(a-82~-19)

550

-

5~15 (M-103-90) 606619(M-2xgO)

M-(a-73))559I

Masssp•ctrum••(a)••3•2•-tri-•-trimcthy•si]y]-N-(2•-hydr•xyst•ar•y])sphin•anineand

, . . . . . . . . . . 350 zOO

1 t31Ll~i2I

362 38~

(M- a-90-19~

"

381

/ (b.2)/[ ~<.-~-~-<~-I))M I l/

(f)

350

339

/ I

(M-a-89-16

~.~c.-~-p(g-,))

~-d) 313

l,, .li ....... . . . . . .~1. . 250 300

(M-(b.l.c))

207 22

7

(~-(b+l.c}

. ..,ll. . ,I..L . ,.............. .... , .... 100 150 200

25 ¸

5o

75,

100-

100

,,

so-

/5-

I00"

'

K

Z

O (")

t-

Z

(3

60

K . S A M U E L S S O N A N D B. S A M U E L S S O N

at the same m/e value in all homologs and ions that shift 28 mass units between adjacent homologs. There are five "molecular weight fragments" which have been considered above and seven "fatty acid fragments" in sphingosine ceramides (fig. 12) viz. ( M - ( a - 7 3 ) ) , ( M - a ) , ( M - a - 1 6 ) , ( M - a - 8 9 ) , (b +1 + 73), (b + 2) and (f) which are formed by elimination of the main part or the whole of the sphingosine carbon skeleton. The "LCB fragments" consist of three ions: ( M - ( b + 1)), m/e 426; ( M - ( b + 1 +90)), m/e 336 and (M - d ) , m/e 311, and are formed by elimination of the fatty acid part of the ceramide. In addition there is an ion at m/e 243, ( M - ( b + 1 +e)), which appears to be specific for sphingosine ceramides as it is not present in sphinganine ceramides. Fig. 13 shows the homologous series of sphinganine ceramides containing 2-hydroxy acids and fig. 14a the mass spectrum of 1,3,2'-tri-O-trimethylsilyl-N-(2'-hydroxystearoyl) sphinganine. Inspection of the latter mass spectrum shows that the molecular weight can be determined from the ions (M), m/e 799; ( M - 15), m/e 784; ( M - 9 0 ) , m/e 709; ( M - 103), m/e 696; ( M - 2 x 90), role 619 and (M -103 - 90), m/e 606. These fragmentations are analogous to those described above for sphingosine ceramides. Some of the "fatty acid fragments" are also analogous to those described above, namely ( M - ( a - 7 3 ) ) , m/e 599; ( M - a ) , m/e 486; ( M - a - 1 6 ) , m/e 470; (b+2), m/e 372; (f), m/e 327; (b + 1 +73), m/e 444. A prominent ion appearing at m/e 397 is considered to be due to ( M - a - 89), see fig. 15. This ion is proba-

-CH=CH2NHI[

-7+

C--CH--(CH2)nCH3

II II

O O

1

Si(CH3)3 Fig. 15. Proposed structure of the ion (M -- a -- 89), m/e 397. bly formed by cleavage between C-2 and C-3 and elimination of the trimethylsilyloxy radical at C-I. Ions due to ( M - ( a - 7 3 ) - 1 6 ) , m/e 543; ( M - a - 8 9 - 1 6 ) , m/e 381 and ( M - ( a - 7 3 ) - 9 0 ) , m/e 469 are considered to be formed from the corresponding ions by elimination of one molecule of methane [cf. 21, 22)] and one molecule of trimethylsilanol, respectively. Reactions leading to "LCB-fragments" (M - (b + 1)) at m/e 428 and (M - d)

SYNTHETIC AND NATURALLY OCCURRING CERAMIDES

61

at m/e 313 have been discussed above for the sphingosine derivatives where similar fragmentations are observed. However, an ion of the same category appearing at m/e 575, ( M - ( g - 1)) has been attributed to rearrangement of a y-hydrogen atom and cleavage of the bond fl to the carbonyl group to give the ion shown in fig. 16. A series of ions has been interpreted to be formed by the B-cleavage described above and in addition cleavage between C-2 and C-3 with transfer o f a trimethylsilyl radical. One of these ions appears at m/e 335 and is assumed R t --CH--CH--CH 2 - O S i ( C H 3 ) 3

RI--CH--CH-C[t2OSi(CH3) 3

I

I

I

O

NH

I

(CH3)3Si

I

C

O

/

I

O

Si(CH3) 3

,

NH

l

I

(CHa)3Si

C

CH

/ O

/ \ /

~--~,\ / 0

Si(CH3) a

-i-O

C,

I

H

Cn

I

R2

Fig. 16. Proposed formation of the ion (M -- (g -- 1)), m/e 575. to be due to ( M - ( a - 7 3 ) - ( g l)). The presence of a metastable peak at the calculated rn/e 200.76 indicates that the initial reaction is a cleavage of the C-C bond between C-2 and C-3 with transfer of the trimethylsilyl radical to the nitrogen. The ion formed in this way is subsequently subjected to fl-cleavage (fig. 17). Ions formed by additional elimination of one or two molecules of trimethylsilanol appear at m/e 245, ( M - ( a - 7 3 ) - ( g - 1 ) - 9 0 ) and at m/e 155 (M - ( a - 73) - ( g - 1 ) - 2 x 90), respectively. An ion appearing at role 217 was earlier observed in the mass spectra of sphinganine ceramides with normal fatty acids. This ion, which was interpreted to be ( M - ( b + l +c)) is also present in sphinganine ceramides containing 2-hydroxy acids. In order to provide additional support for the proposed fragmentations 1,3,2'-d2v-tri-O-trimethylsilyl-N-(2'-hydroxystearoyl) sphingosine was prepared using dg-TMCS 18). The mass spectra of corresponding nondeuterated and deuterated ceramide derivatives are shown in figs. 10a and 10b. The presence of three intact TMS groups is shown by the shift of the molecular ion by 27 mass units, from m/e 797 in fig. 10a to role 824 in fig. 10b. Loss of

62

K. SAMUELSSONAND B. SAMUELSSON

methyl radical f r o m a T M S g r o u p is confirmed by the shift o f ( M - 15) f r o m

m/e 782 to m/e 806. The ions f o r m e d in the reactions discussed a b o v e for sphingosine ceramides can be divided into three groups, i.e. those c o n t a i n i n g one, two or three T M S groups p e r molecule. The results show that there is c o m p l e t e agreement between the p r o p o s e d n u m b e r o f T M S g r o u p s in the

RI-CH

7- CH--CH2OSi(CH3) 3

Ij

I

O

+ NH

/ Si(CH3) 3

I-J'l

(CH3) 3 Si

C

0

~ \ / 0

Ca

H

CH 2

I

\/ C.

I R2

•CH--CH2OSi(CH3) 3

I

(CHa) a S i - +NH

Si(CH3) 3

I

C

O

/

~/._.,~ / O

CH

Cn

I

R2 "CH--CH2OSi(CH3)3

I

----* (CH3)3Si-- +NH

Si(CH3)3

I

C

O

/

/ \ / O

CH

J

H

Fig. 17. Proposed formation of the ion (M -- (a -- 73) -- (g -- 1)),

m/e 335.

SYNTHETIC AND NATURALLY OCCURRING CERAMIDES

63

ions and the Am/e between the deuterated and nondeuterated derivatives. Of specific interest is the intramolecular transfer reaction of a trimethylsilyl group occurring during the formation of (M - ( a - 73)) and (b + 1 + 73). This reaction is strongly supported by the observed shifts of 27 mass units for the former ion and 18 mass units for (b + 1 +73), demonstrating the presence of three and two TMS groups, respectively. The ion ( M - a - 1 6 ) at m/e 470 shifts 15 mass units and is considered to be formed from ( M - a) by elimination of one TMS methyl group as methane after rearrangement of a hydrogen atom 21, 22). Deuterated derivatives of sphinganine ceramides were also prepared. Mass spectra of 1,3,2'-d27-tri-O-trimethylsilyl-N-(2'-hydroxystearoyl) sphinganine and 1,3,2'-tri-O-trimethylsilyl-N-(2'-hydroxystearoyl) sphinganine are shown in fig. 14. Conclusions similar to those described above can be drawn from these data. Additional support for the intramolecular transfer of a TMS TABLE 4

Major ions in mass spectra of ceramides. (Relative abundancies given are for the C18-fatty acid ceramides) Sphingosine

LCB

Fatty acid

Normal

Sphinganine

2-hydroxy

Normal

M

1%

1%

1%

M--15

7

4

8

M

90

2-hydroxy

2% 11

5

6

5

5

M - - 103

3

3

13

13

M--2

2

4

1

2

M - - 103 - - 9 0 M a

×90

4

9

100

100

8 16

1 25

M--a--16 M - - ( a - - 73)

20

13 19

33

100

M - - ( a - - 73) - - 16

-

3

-

8

M - - ( a - - 73) - - 9 0 M -- a -- 89

-

3 6

44

8 86

M--a--89--16 M - - ( a - - 73) - - ( g - - 1)

7

1 3

15

5 14

M - - ( a - - 73) - - ( g - - 1) - - 9 0

23

6

62

17

M - - ( a - - 73) - - ( g - - 1) - - 2 × 9 0 M - - ( b + 1)

45

3 22

4

8 5

M (b÷1+90) M--(b÷ 1 +e)

6 24

10 12

-

-

M--(b+

1 +c)

b÷l +73 b +2 M --d f M --(g--

1)

8

-

4

28

30

4 60

14 5 31

5 I00

24 20 58

-

20

-

30

-

-

-

5

64

K . S A M U E L S S O N A N D B. S A M U E L S S O N

group is derived from shifts of 27, 18 and 9 mass units, respectively, for (M - ( a - 7 3 ) - ( g - 1)), (M - ( a - 7 3 ) - ( g - 1)-90) and ( M - ( a - 7 3 ) - ( g - 1) - 2 × 90). The ions ( M - ( a - 7 3 ) - 16), m/e 543 and ( M - a - 8 9 - 16), m/e 381 show shifts of 24 mass units and 6 mass units, respectively, and are probably formed in the same way as ( M - a - 16) (cf. above). Table 4 shows the occurrence of mass spectral ions in sphingosine and sphinganine ceramides containing normal and 2-hydroxy acids. There are several ions that occur in all the ceramides listed, namely the "molecular weight fragments" (M), (M-15), (M-90), ( M - 2 × 9 0 ) , (M-103), ( M 103-90); the "fatty acid fragments" ( M - a ) , ( M - ( a - 7 3 ) ) , ( b + l +73), (M-(a-73)-(g-l)), (M-(a-V3)-(g-I)-90) and the "LCB fragments" ( M - d), ( M - (b +1)). The ion ( M - ( b + 1 +e)) at m/e 243 seems to be specific for ceramides containing sphingosine as LCB. The presence of a double bond between C-4 and C-5 is probably required for the elimination of (e). With sphinganine or 4-hydroxysphinganine (phytosphingosine)2a) as LCB, ( M - ( b + l +c)) appears instead of ( M - ( b + I +e)). The presence of a 2'-hydroxyl group in the ceramides causes cleavage of the C-C bond between C-I' and C-2' with formation of the ion (f). This ion is fairly abundant and is specific for 2'-hydroxy ceramides. The "molecular weight fragments" and the "fatty acid fragments" appear at m/e values which are 88 mass units higher in ceramides containing 2'-hydroxy acids than in the normal fatty acid analogs, because of the extra trimethylsilyloxy group. The "LCB fragments", with the exception of ( M - ( g - 1 ) ) , do not show this shift. The latter ion retains C-2'. Separation and identification of ceramides derived from human plasma sphingomyelins 24) Previous studies have shown that sphingomyelins, when subjected to thinlayer chromatography (TLC), give rise to two spots with preferentially longchain fatty acids in the faster-moving fraction 2~ 28). Similar fractionations have been observed in chromatography on silicic acid29). It has also been demonstrated recently that ceramide acetates derived from human serum sphingomyelins can be separated. Three fractions obtained in this way were analyzed by gas-liquid chromatography (GLC) after hydrolysis and were shown to differ with respect to the fatty acid patterna°). In early studies Sweeley and Moscatelli 1) analyzed the LCB of plasma sphingomyelins by periodate oxidation and GLC of the resulting aldehydes. This investigation revealed the presence of a sphingadienine base in addition to sphing-4-enine and sphinganine. Later the sphingadienine was reported to consist of a mixture of sphinga-4,14-dienine and sphinga-4,12-dienine3X). More detailed chemical studies established that the doubly unsaturated

SYNTHETIC AND NATURALLY

OCCURRING

CERAMIDES

65

base is sphinga-4,14-dienine4,6). Bases with shorter chain length, i.e., hexadecasphing-4-enine and heptadecasphing-4-enine 1,31, 6, 2) were also identified, as well as trace amounts of hexadecasphinganine and heptadecasphinganine 31). Analyses of fatty acids from human plasma sphingomyelins32,8, 33-a5) have shown the presence of all even- and odd-carbon saturated fatty acids from Cl0 to C26. Palmitic acid was the predominant component of these acids and 18:0, 20:0, 22:0, 23:0 and 24:0 were present in relatively large amounts. The monoenoic acid 24:1 was one of the major acids, whereas other monoenoic acids were present only in small amounts. Svennerholm and coworkers, however, have found a higher percentage of 22: l and 24: 1 than other investigators34). Small amounts of polyunsaturated fatty acids have also been reported 32, 8, zz, 35). If each of the bases were combined with each of the fatty acids known to occur in sphingomyelins it is evident that a large number of molecular species of ceramides could exist. These considerations indicate that efficient methods are required for the separation and identification of individual molecules of sphingomyelins. An approach to methods for analysis of sphingomyelins with respect to molecular species of derived ceramides was recently carried out. The procedure was based on fractionation of ceramide diacetates according to degree of unsaturation and also to some extent according to chain length. Ceramides were subsequently separated by GLC according to the number of carbon atoms after conversion to di-O-trimethylsilyl ether derivatives. Molecular species were identified by GLC-mass spectrometry z0). The sphingomyelins isolated by silic acid chromatography after mild alkaline methanolysis of the lipid extract of human plasma were hydrolyzed enzymatically with phospholipase C. The ceramides were acetylated in order to improve the separation by TLC on silica gel containing silver nitrate. The reference compounds N-stearoyl sphingosine diacetate, N-oleoyl sphingosine diacetate, and N-linoeoyl sphingosine diacetate had Rf values of 0.75, 0.44, and 0.23, respectively. There was no significant separation between corresponding sphinganine and sphingosine ceramide derivatives (fig. 18). In order to test the effect of the chain length of the fatty acids on the thin-layer chromatographic separation, we prepared and analyzed additional ceramide acetates. It was found that with saturated fatty acid derivatives the chain length had little influence on the mobility (N-lignoceroyl sphingosine diacetate, Rf 0.78 and N-stearoyl sphingosine diacetate, Rf 0.75). On the other hand, the mobility of ceramide diacetates containing cis monounsaturated fatty acids was greatly influenced by the chain length of the fatty acid (fig. 19) (N-cis-nervonoyl sphingosine diacetate, Rf 0.60, and

K. SAMUELSSONAND B.SAMUELSSON

66

,,,

DIH 1@:I

t

@

O

T!~

j ,m.

dPH 18t0

37H 1B: 1

T/ '(

I

SPH 18m2

Fig. 18. Thin-layer chromatogram of acetylated ceramides derived from human plasma sphingomyelins. Adsorbent : Silica Gel G: silver nitrate 15/1. Solvent: chloroform/benzene/methanol 80/20/1. References: N-stearoyl-sphinganine diacetate (DIH 18:0), N-oleoylsphinganine diacetate (DIH 18:1), N-linoleoyl-sphinganine diacetate (DIH 18:2), Nstearoyl-sphingosine diacetate (SPH 18:0), N-oleoyl-sphingosine diacetate (SPH 18: 1) and N-linoleoyl-sphingosine diacetate (SPH 18: 2). N-oleoyl sphingosine diacetate Rf 0.44). An intermediate effect of the chain length was observed with trans monounsaturated fatty acid derivatives (Ntrans-nervonoyl sphingosine diacetate Rf 0.68, and N-elaidoyl sphingosine diacetate, Rf 0.60). The T L C of the ceramide diacetates derived from human plasma is shown in fig. 18. For practical purposes the separated material was divided into four fractions. Fraction I had a mobility corresponding to that observed for saturated fatty acid ceramide diacetates. Fraction II had approximately the same Rf value as N-cis-nervonoyl sphingosine diacetate, and fraction III appeared between that compound and N-oleoyl sphingosine diacetate. Fraction IV appeared as a well-defined band slightly below the last reference compounds. The isolated fractions were subjected to methanolysis of the O-acetyl esters and converted into trimethylsilyl ether derivatives. These were further separated by G L C on 1 ~o OV-1 at 270°C. The gas chromatogram of fraction I

SYNTHETIC AND NATURALLY OCCURRING CERAMIDES

67

!l,~ A

!

18:0

24:1 tr 24:1

cis l

O

24:1 cis I

|

18:1 cis

Fig. 19. Thin-layer chromatogram of diacetates of synthetic ceramides. N-stearoylsphingosine diacetate (18:0), N-nervonoyl-sphingosine diacetate (24:1 cis), N-transnervonoyl-sphingosine diacetate (24: 1 trans) and N-oleoyl-sphingosine diacetate (18: 1 cis).

68

K . S A M U E L S S O N A N D B. S A M U E L S S O N

6-

5txJ tO Z /4O ck to tlJ

3-

1:3

1:1

rr

o

tJ

2"

1:4

t.O

1-

6

4

8 10 12 MINUTES

14

1'8 2'0

16

Fig. 20. Gas-liquid c h r o m a t o g r a p h y o f ceramides in fraction I of fig. 18. T h e acetates were hydrolyzed a n d converted to trimethylsilyl ether derivatives. C o l u m n : 1 ~ OV-1. T e m p e r a t u r e s : c o l u m n , 275°C, flash heater: 300°C, detector 300°C. Carrier gas: helium. TABLE 5

M a s s spectrometric data for di-O-trimethylsilyl (TMS) ether derivatives of ceramides f r o m T L C fraction I (fig. 20). T h e designation of m a s s spectrographic f r a g m e n t s is s h o w n below C17H35

I CO

I OTMS

I CHz(CH2)12--CH--CH--CH

NH

I -- C H - - C H 2 - - O T M S

or --CH2

d CH2 --

a

GLC fraction* T G C U

I:1

35.4

LCB fragments M --d

m/e

~

285 283 299 297 313 311 309

1 28 2 3 100 1

LCB

16:0 16:1 17:0 17:1 18:0 18:1 18:2

F a t t y acid fragments M -- a

m/e

°/o

342 340 356 354 370 368

12 1 20 3

Fatty acid

Main constituent(s)

14:0 14:1 15:0 15:1 16:0 16:1

LCB 18:1-14:0 LCB 16:1-16:0

S Y N T H E T I C A N D N A T U R A L L Y O C C U R R I N G CERAMIDES

69

Table 5 (continued)

GLC fraction* TGCU

1:3

1:5

1:7

1:9

I:11

* + § '

37.3

39.4

41.3

43.3

45.3

LCB fragments M -- d

m/e

~

285 283 299 297 313 311 309 285 283 299 297 313 311 309 285 283 299 297 313 311 309 285 283 299 297 313 311 309 285 283 299 297 313 311 309

3 1 2 89 2 62 2 3 3 100 5 4 100 1 5 2 93 5 1 8 2 4 100 2 2 5 5 3 100 2

LCB

Fatty acid fragments M -- a

Fatty acid

Main constituent(s)

m/e 16:0 16:1 17:0 17:1 18:0 18:1 18:2 16:0 16:1 17:0 17:1 18:0 18:1 18:2 16:0 16:1 17:0 17:1 18:0 18:1 18:2 16:0 16:1 17:0 17:1 18:0 18:1 18:2 16:0 16:1 17:0 17:1 18:0 18:1 18:2

370 368 384 382 398 + 396

100 2 3 -

16:0 16:1 17:0 17:1 18:0 18:1

LCB 18:1-16:0 LCB 16:1-18:0

398§ 396 412 410 426§ 424§

77 3 2 47 2

18:0 18:1 19:0 19:1 20:0 20:1

LCB18:1-18:0 LCB 16:1-20:0

426' 424' 440 438 454 452

33 2 2 5 60 11

20:0 20:1 21:0 21:1 22:0 22:1

LCB18:1-20:0 LCB 16:1-22:0 LCB16:1-22:l

454 452 468 466 482 480

54 21 1 1 5 3

22:0 22:1 23:0 23:1 24:0 24:1

LCB 18:1-22:0 LCB18:1-22:1 LCB 16:1-24:0 LCB 16:1-24:1

482 480 496 494 510 508

21 33 -

24:0 24:1 25:0 25:1 26:0 26:1

LCB18:1-24:0 LCB18:1-24:1

Designation of GLC fractions corresponds to that used in fig. 20. 398: LCB and fatty acid fragment. 398, 424, 426: LCB and fatty acid fragments. 424 and 426: LCB and fatty acid fragments.

is s h o w n i n fig. 20. A t l e a s t e l e v e n d i f f e r e n t p e a k s c o u l d b e r e c o g n i z e d . P e a k s I : 3 , 1:5, I : 7 , 1:9 a n d I : 1 1 h a d r e t e n t i o n t i m e s p r a c t i c a l l y e q u a l t o those obtained for the same derivative of sphingosine ceramides with the f a t t y a c i d s 1 6 : 0 , 18:0, 2 0 : 0 , 2 2 : 0 a n d 2 4 : 0 , r e s p e c t i v e l y ( t a b l e s 2 a n d 5).

70

K . S A M U E L S S O N A N D B. S A M U E L S S O N

In a separate run fraction I was analyzed by the combined gas chromatograph-mass spectrometer which gave an equivalent gas chromatogram. Mass spectra were recorded on some of the peaks. The mass spectrum of the gas chromatographic fraction l : l showed that the LCB fragments M - d appeared at m/e 283 (28%) and at m/e 311 (100%). These ions are due to hexadecasphing-4-enine and sphingosine, respectively (table 5). The fatty acid fragments M - a had m/e values of 342 (12~o) and 370 (20~o), which showed the presence of the fatty acid residues 14:0 and 16: 0. The retention time of I: 1 ( T G C U 35.4) was essentially the same as that expected for the same derivative of N-myristoyl sphingosine ( T G C U 35.5) (tables 2 and 5). On the basis of the G L C data and the mass spectrometric analysis we concluded that one of the molecular species is LCB 18: 1-14:0. However, the mass spectrometric data also showed the presence of the 16: 1 LCB and the fatty acid residue 16: 0. Since it is expected that the gas chromatographic separation on the column is primarily determined by the total number of carbon atoms in the ceramides, another constituent in this fraction must be LCB 16: 1-16:0. Low intensity ions, which may be due to the LCB sphinganine, hexadecasphinganine, and heptadecasphing-4-enine were also observed. These LCB should be combined with the fatty acids 14:0, 16:0, and 15:0, respectively. Other gas chromatographic fractions derived from material in T L C fraction I were analyzed in a similar manner and the main constituents are

6"

5-

,,,4I.o z

IT:11

o

If) ILl n~

3-

o::

,,o ~j

11:2

;;:9

11:1

i

2

4

6

8

10

12

i

1'~ 16 18 2'0

MINUTES

Fig. 21. Gas-liquid chromatography of ceramides in fraction II of fig. 18. For details see fig. 20.

300

350 '

3~o

(M-(b+l÷90))

(M-d)

6

3 0

336 Ji, ,,~ ......

(M-(b+l÷90))

311

(M-d)

6

4 450 '

460

.~.

438463

450

(M-(b+l)) 426

4 0

.k

(M-(b+l)) 421a~ 6

6

5 0

~6o

(M-a) 480

480

(M-a)

.

~. . 600 .

5so

(M-(a-73)) 553

a

660

(CH2)-~I~CH:

550 .

553 L

(M-(a -73))

b

700

C:O

~

6~o

700

(M-90) (M-103) 701

d

7~o

(M-15)

860 m/~

BOOm/e '

\l.. 791

(M-15) 776 (M)

7~ 0

(?H2)]3 CH= CH (CH2)7 CH 3

650

H, CH-CH2-OTMS

.

.

(M-103) 688 (M-90) ~ 701 . . ~ L

Mass spectra o f di-O-trimethylsilylether derivatives of G L C fraction II: l 1 o f fig. 21 (A) and o f N-nervonoyl-sphingosine (B).

250

247

243

(M-(b.l.e))

B

250 '

~//

,,

[/247 258

(M-(b+1+e)) 2~3

A

Fig. 22.

200

. 25

uJ

~ 50

Z

tu 75, (J z

100

200

ILl > I-< ~j 25" e,"

Z m 50. <

hl 75' (J Z

100

-.J

>.

ffl

0 ('1

C

Z

C~ > Z

72

K . S A M U E L S S O N A N D B. S A M U E L S S O N

g i v e n in t a b l e 5. T h e L C B c o n s i s t e d m a i n l y o f s p h i n g o s i n e a n d h e x a d e c a s p h i n g - 4 - e n i n e c o m b i n e d in each f r a c t i o n w i t h s a t u r a t e d fatty acids differing in c h a i n l e n g t h by t w o c a r b o n a t o m s . H o w e v e r , the gas c h r o m a t o g r a p h i c f r a c t i o n s I : 7, I: 9, a n d I: 11 also c o n t a i n e d the f a t t y a c i d residues 22: 1 a n d 24: 1. In v i e w o f the m o b i l i t y o f f r a c t i o n i o n T L C (fig. 18) c o m p a r e d w i t h t h e r e f e r e n c e c e r a m i d e L C B 1 8 : 1 - 2 4 : 1 (cis) (fig. 19) it seems unlikely t h a t t h e s e c e r a m i d e s c o n t a i n (n-9) cis m o n o u n s a t u r a t e d

fatty acids. F u r t h e r

studies are r e q u i r e d to establish the n a t u r e o f t h e m o n o u n s a t u r a t e d

fatty

acids in this f r a c t i o n . T L C f r a c t i o n II (fig. 21) c o n t a i n e d m a i n l y c e r a m i d e s w i t h l o n g - c h a i n f a t t y acids (for G L C s e p a r a t i o n see fig. 22), n a m e l y 20: 1, 22: 1, a n d 24:1 a n d the LCB sphingosine and hexadecasphing-4-enine. Mass spectrometric data for t h e m a i n c o n s t i t u e n t s are g i v e n in t a b l e 6. It is w o r t h n o t i n g t h a t the m a i n p e a k (II : 11) o f f r a c t i o n 11 is a l m o s t exclusively d u e to the m o l e c u l a r species TABLE 6

Mass spectrometric data for di-O-trimethylsilyl ether derivatives of ceramides from TLC fraction II (fig. 18). Designation of mass spectrographic fragments is shown in table 5

GLC fraction* T G C U

11:7

11:9

1I:11

41.3

43.3

45.3

LCB fragments M-- d m/e

~

285 283 299 297 313 311 309 285 283 299 297 313 311 309 285 283 299 297 313 311 309

6 100 1 3 5 58 9 28 3 1 100 6 100 2

LCB

16:0 16:1 17:0 17:1 18:0 18:1 18:2 16:0 16:1 17:0 17:1 18:0 18:1 18:2 16:0 16:1 17:0 17:1 18:0 18:1 18:2

Fatty acid fragments M--a

Fatty Main acid constituent(s)

rn/e

°/o

426 + 424 + 440 438 454 452

4 5 3 20

20:0 20:1 21:0 21:1 22:0 22:1

454 452 468 466 482 480

4 15 1 19

22:0 22:1 23:0 23:1 24:0 24:1

482 480 496 494 510 508

86 -

24:0 24:1 25:0 25:1 26:0 26:1

* Designation of GLC fractions corresponds to that used in fig. 21. + 424 and 426: LCB and fatty acid fragments.

LCB 18:1-20:1

LCB 16:1-22:1

LCB18:1-22:1 LCB 16:1-24:1

LCB 18:1-24:1

SYNTHETIC AND NATURALLY OCCURRING CERAMIDES

73

w z

/4w n--

3Oc o

m:3

2

Fig. 23.

4

6

8 10 12 MINUTES

14

16

18

20

Gas-liquid chromatography of ceramides in fraction III of fig. 18. For details see fig. 20.

W O U') U.I r,,. 3 " rr

o z.W

IV: 11

2'o MINUTES

Fig. 24.

Gas-liquid chromatography of ceramides in fraction IV of fig. 18. For details see fig. 20.

74

K. SAMUELSSON A N D B. SAMUELSSON

L C B 18: 1 - 2 4 : 1. F o r c o m p a r i s o n , t h e m a s s s p e c t r a o f I I : 11 a n d s y n t h e t i c L C B 18: 1 - 2 4 : 1 a r e g i v e n in fig. 22. T L C f r a c t i o n I I I (fig. 18) w a s a m i n o r c o m p o n e n t a n d c o n s i s t e d o f a r e l a t i v e l y d i f f u s e b a n d . T h e G L C s e p a r a t i o n o f t h i s m a t e r i a l (fig. 23) s h o w e d s e v e r a l p e a k s , o f w h i c h 111:9 p r e d o m i n a t e d .

It h a d t h e s a m e r e t e n t i o n

t i m e ( T G C U 43.5) as t h a t ( T G C U 43.5) f o u n d f o r L C B 1 8 : 1 - 2 2 : 0 ( t a b l e s 2 a n d 7). T h e G L C - m a s s

s p e c t r o m e t r i c a n a l y s e s a r e s u m m a r i z e d in t a b l e 7.

All o f t h e p e a k s a n a l y z e d c o n t a i n e d s p h i n g a - 4 , 1 4 - d i e n i n e . T h e f i n d i n g t h a t TABLE 7 Mass spectrometric data for di-O-trimethylsilyl ether derivatives of ceramides from TLC fraction lII (fig. 18). Designation of mass spectrographic fragments is shown in table 5

GLC fraction* TGCU

LCB fragments M -- d

m/e III:3

II1:5

111:7

111:9

37.5

39.5

41.5

43.5

285 283 299 297 313 311 309 285 283 299 297 313 311 309 285 283 299 297 313 311 309 285 283 299 297 313 311 309

LCB

%o 3 3 13 100 27 3 13 100 16 1 16 100 12 1 2 35 100

16:0 16:1 17:0 17:1 18:0 18:1 18:2 16:0 16:1 17:0 17:1 18:0 18:1 18:2 16:0 16:1 17:0 17:1 18:0 18:1 18:2 16:0 16:1 17:0 17:1 18:0 18:1 18:2

Fatty acid fragments M -- a

Fatty acid

Main constituent(s)

LCB18:2-16:0 LCB18:1-16:1

rn/e

%

370 368 384 382 398 396

74 4 -

16:0 16:1 17:0 17:1 18:0 18:1

398 + 396 412 410 426 + 424 +

41 4 1 10

18:0 18:1 19:0 19:1 20:0 20:1

426§ 424§ 440 438 454 452

9 3 2

20:0 20:1 21:0 21:1 22:0 22:1

454 452 468 466 482 480

45 23 1 1 8

22:0 22:1 23:0 23:1 24:0 24:1

* Designation of GLC fractions corresponds to that used in fig. 23. + 398, 424, 426: LCB and fatty acid fragments. § 424 and 426: LCB and fatty acid fragments.

LCB 18:2-18:0 LCB 18:1-18:1 LCB 16:1-20:1

LCB 18:2-20:0 LCB18:1-20:1 LCB 16:1-22:1

LCB 18:2-22:0 LCB18:1-22:1 LCB16:1-24:1

SYNTHETIC AND NATURALLY OCCURRING CERAMIDES

75

T L C fraction IV (see below) consisted of sphinga-4,14-dienine ceramides with m o n o u n s a t u r a t e d fatty acids indicates that the sphinga-4,14-dienine ceramides of fraction III c o n t a i n m a i n l y saturated fatty acids. The occurrence in fraction I I I of ceramides with the LCB sphingosine a n d hexadecasphing-4enine a n d m o n o u n s a t u r a t e d fatty acids is also evident from table 7. Some of these ceramides, i.e., LCB 18:1-22:1 a n d LCB 1 8 : 1 - 2 0 : 1, appear to be c o n t a m i n a n t s from fraction I1. T L C fraction IV appeared as a well-defined b a n d which o n G L C gave two m a i n peaks (fig. 24). These were almost exclusively due to the molecular species LCB 18: 2-22: 1 a n d LCB 18 : 2-24: 1, respectively (table 8). TABLE 8

Mass spectrometric data for di-O-trimethylsilyl ether derivatives of ceramides from TLC fraction IV (fig. 18). Designation of mass spectrographic fragments is shown in table 5

GLC fraction* TGCU

IV:9

43.4

IV:ll

45.3

LCB fragments M -- d

m/e

%

285 283 299 297 313 311 309 285 283 299 297 313 311 309

3 2 1 100 1 2 1 2 1 100

LCB

Fatty acid fragments M -- a

Fatty acid

Main constituent(s)

m/e 16:0 16:1 17:0 17:1 18:0 18:1 18:2 16:0 16:1 17:0 17:1 18:0 18:1 18:2

454 452 468 466 482 480

5 -

22:0 22:1 23:0 LCB 18:2-22:1 23:1 24:0 24:1

482 480 496 494 510 508

69 -

24:0 24:1 25:0 LCB 18:2-24:1 25:1 26:0 26:1

* Designation of GLC fractions corresponds to that used in fig. 24.

Free ceramides in human plasma Ceramides have been shown to act as precursors in the biosynthesisa6-a8) of sphingolipids a n d they have also been d e m o n s t r a t e d to be intermediates in the degradation an,4°) of this group of c o m p o u n d s . I n view of these findings it seemed of particular interest to establish if free ceramides are present in blood plasma. H u m a n plasma was extracted with c h l o r o f o r m - m e t h a n o l 41) a n d the lipid extract after a d d i t i o n of N-(1-14C)oleoyl-sphinganine was fractionated o n silicic acid. The material eluted with c h l o r o f o r m - m e t h a n o l (95:5, v/v) was

76

K. SAMUELSSON

AND

B. SAMUELSSON

further chromatographed on silicic acid. The column was eluted with ethyl acetate-benzene (10: 90, v/v) followed by ethyl acetate-benzene (40: 60, v/v). The material in the second fraction (85 ~o of added 14C) was separated by thin-layer chromatography on silica gel G. The plates were sprayed with 2,7-dichlorofluorescein in ethanol, and the material in the zone corresponding to the reference, N-oleoyl-sphinganine (Rf 0.3), was isolated. This material was acetylated and separated by argentation chromatography. Two fractions were isolated, viz. I (Rf 0.614).78) and II (Rf 0.51-0.61). The references had the following Rf values, N-stearoyl-sphingosine diacetate, 0.75; N-nervonoylsphingosine diacetate, 0.60; and N-oleoyl-sphingosine diacetate, 0.44. The fractions were subjected to mild alkali-catalyzed methanolysis a) and converted to trimethylsilyl ether derivatives z) before analysis by gas chromatography and gas chromatography-mass spectrometry (table 9). Separation of fraction I by gas chromatography (fig. 25A) showed eleven components. Analysis of fraction I:3 by mass spectrometry in combination with gas-liquid chromatography (table 9) demonstrated that the principal long-chain base was sphingosine ( M - d , m/e 31 ]) and that the main fatty acid residue was palmitic acid ( M - a, m/e 370). That these compounds were LU U3 Z 0 0.. U') LU n,- 2 . n,-

i6

,:9

A

U J,I Q

"Ltl I / [:5 / ]:?

i

i

~ i

A

I:~1

1'o 17 i~ 17 1'8 io 2? MINUTES

taJ tt) z

o

o.. ~2-

B

~:2 l:l/

~':ll

D1W !

MINUTES

Fig 25. Gas chromatographic separation of di-O-trimethylsilyl ether derivatives of ceramides from human plasma. (A) Thin-layer chromatographic fraction I. (B) Thin-layer chromatographic fraction II.

77

SYNTHETIC AND NATURALLY OCCURRING CERAMIDES TABLE 9

Retention times and mass spectrometric data for di-O-trimethylsilyl ether derivatives of plasma and reference ceramides OTMS

NHCOR

I

I

CHa(CHDn--CH=CH--CH -- CH--CH2--O--TMS or

--CH2--CH2-+

a

÷ +-------

Triglyceride

material

carbon units

m/e

%o

m/e

%

37.5 37.5 39.4 39.4 41.4 41.4 43.3 43.5 44.3 44.5 45.2 45.5 43.1 43.3 45.1 45.3

311 311 311 311 311 311 311 311 311

90 72 97 61 100 38 100 65 100

370 370 398 398 426 426 454 454 468

100 100 100 100 51 100 94 100 48

666 666 694 694 722 722 750 750 764

7 6 9 7 5 5 8 6 7

311 311 311

100 72 100

482 482 452

78 100 25

778 778 748

2 LCB 18:1-24:0 6 1 LCB 18:1-22:1

311 311

95 71

480 480

100 100

776 776

6 LCB 18:1-24:1 6

18:1-16:0 18:1-18:0 18:1-20:0 18:1-22:0 18:1-23:0 18:1-24:0 18:1-22:1 18:1-24:1

M -- a

- - +

Analyzed

I:3 LCB I:5 LCB I:7 LCB I:9 LCB I:10 LCB I'll LCB II:9 LCB II:ll LCB

M -- d

d

M--15

Main

m/e %o constituent LCB 18:1-16:0 LCB 18:1-18:0 LCB 18:1-20:0 LCB 18:1-22:0 LCB 18:1-23:0

c o m b i n e d to give the m o l e c u l a r species N - p a l m i t o y l - s p h i n g o s i n e was app a r e n t f r o m the f r a g m e n t M-15 which a p p e a r e d at

m/e 666. Th e retention

time o f fraction I : 3 was also identical with that f o u n d for the reference N - p a l m i t o y l - s p h i n g o s i n e (LCB 1 8 : 1 - 1 6 : 0 ) . The other fractions were analyzed in a similar way, and the main constituents f o u n d are given in table 9. It should also be n o t e d that the mass spectrometric d a t a indicated the

m/e 313) and hexadecasphingm/e 283) as long-chain base in each fraction. Th e ceramides

presence o f ceramides with sphinganine (M - d, 4-enine ( M - d ,

with the latter long-chain base c o n t a i n e d a fatty acid with two m o r e c a r b o n a t o m s t h a n the sphingosine a n d sphinganine ceramides o f the same fractions. Thin-layer c h r o m a t o g r a p h i c fraction II was also analyzed by gas chrom a t o g r a p h y (fig. 25B) a n d the two m a i n c o m p o n e n t s , I I : 9 an d I I : 11, were f u r t h e r studied by mass s p e c t r o m e t r y (table 9). T h e m a i n co n st i t u en t o f

78

K. SAMUELSSON AND B. SAMUELSSON

fraction I I : 9

was the trimethylsilyl ether derivative o f N - d o c o s e n o y l -

sphingosine and fraction II: 11 was almost exclusively due to the trimethylsilyl ether derivative o f N-tetracosenoyl-sphingosine. Th e mass spectra rec o r d e d on fraction I I : 11 and on the trimethylsilyl ether derivative o f synthetically p r e p a r e d N - n e r v o n o y l - D L - s p h i n g o s i n e were practically identical. This investigation d e m o n s t r a t e s the occurrence and nature o f ceramides in h u m a n plasma. A d d i t i o n a l studies are required to establish their origin and m e t a b o l i c fate. T h e w o r k described also provides a basis for studies o f ceramides in plasma in diseases affecting the m e t a b o l i s m o f sphingolipids.

References 1) 2) 3) 4) 5) 6) 7) 8) 9) 10)

C. C. Sweeley and E. A. Moscatelli, J. Lipid Res. 1 (1959) 40 R. C. Gaver and C. C. Sweeley, J. Am. Oil Chem. Soc. 42 (1965) 294 K. A. Karlsson and G. A. L. Holm, Acta Chem. Scand. 19 (1965) 2423 K. A. Karlsson, Acta Chem. Scand. 21 (1967) 2577 H. E. Carter and R. C. Gaver, Biochem. Biophys. Res. Commun. 29 (1967) 866 A. J. Polito, T. Akita and C. C. Sweeley, Biochemistry 7 (1968) 2609 H. E. Carter, P. Johnson and E. J. Weber, Ann. Rev. Biochem. 34 (1965) 109 C. C. Sweeley, J. Lipid Res. 4 (1963) 402 Y. Kishimoto and N. S. Radin, J. Lipid Res. 4 (1963) 437 J. S. O'Brien and G. Rouser, J. Lipid Res. 5 (1964) 339 11) S. Stallberg-Stenhagen and L. Svennerholm, J. Lipid Res. 6 (1965) 146 12) B. Samuelsson and K. Samuelsson, Biochim. Biophys. Acta 164 (1968) 121 13) K. Samuelsson, to be published 14) C. Litchfield, R. D. Harlow and R. Reiser, J. Am. Oil Chemists' Soc. 42 (1965) 849 15) L. S. Ettre and F. J. Kabot, J. Chromatography 11 (1963) 114 16) J. L. Moore, T. Richardson and C. H. Amundson, J. Gas Chromatography 2 (1964) 318 17) S. M. Grundy, E. H. Ahrens, Jr. and T. A. Miettinen, J. Lipid Res. 6 (1965) 397 18) B. Samuelsson and K. Samuelsson, J. Lipid Res. 10 (1969) 41 19) R. C. Gaver and C. C. Sweeley, J. Am. Chem. Soc. 88 (1966) 3643 20) S. Hammarstr6m, B. Samuelsson and K. Samuelsson, J. Lipid Res, 11 (1970) 150 21) J. A. Mc Closkey, R. N. Stillwell and A. M. Lawson, Anal. Chem. 40 (1968) 233 22) G.H. Draffan, R. N. Stillwell and J. A. Mc Closkey, Org. Mass Spectrom. 1 (1968) 669 23) S. Hammarstr6m, to be published 24) B. Samuelsson and K. Samuelsson, J. Lipid Res. 10 (1969) 47 25) H. Pilz and H. Jatzkewitz, Naturwissenschaften 51 (1964) 61 26) H. Pilz and H. Jatzkewitz, J. Neurochem. 11 (1964) 603 27) P. D. S. Wood, K. Imaichi, J. Knowles, K. Michaels and U Kinsell, J. Lipid Res. 5 (1964) 225 28) P. D. S. Wood and S. Holton, Proc. Soc. Exptl. Biol. Med. 115 (1964) 990 29) K. A. Karlsson, Biochem. J. 92 (1964) 39P. 30) O. Renkonen, J. Am. Oil Chem. Soc. 42 (1965) 298 31) K. A. Karlsson, Acta Chem. Scand. 18 (1964) 2395 32) D. J. Hanahan, R. M. Watts and D. Pappajohn, J. Lipid Res. 1 (1960) 421 33) J. H. Williams, M. Kuchmak and R. F. Witter, Lipids 1 (1966) 89 34) E. Svennerholm, S. St~illberg-Stenhagen and L. Svennerholm, Biochim. Biophys. Acta 125 (1966) 60 35) G. B. Phillips and J. T. Dodge, J. Lipid Res. 8 (1967) 676

SYNTHETIC

36) 37) 38) 39) 40) 41)

AND NATURALLY

OCCURRING

CERAMIDES

S. Basu, B. Kaufman and S. Roseman, J. Biol. Chem. 243 (1968) 5802 P. Morel1 and N. S. Radin, Biochemistry 8 (1969) 506 M. Sribney and E. P. Kennedy, J. Biol. Chem. 233 (1958) 1315 P. B. Schneider and E. P. Kennedy, J. Lipid Res. 9 (1968) 58 Z. Leibovitz and S. Gatt, Biochim. Biophys. Acta 152 (1968) 136 G. B. Phillips, Biochim. Biophys. Acta 164 (1958) 421

79