structure and origin of artifacts in the analysis of plasmalogens

JOURNAL OF MASS SPECTROMETRY, VOL. 31, 303-308 (1996)

Structure and Origin of Artifacts in the Analysis of Plasmalogens*

Angela Dudda and Gerhard Spitellert Institut fur Organische Chemie I, Universitat Bayreuth, Universitatsstrasse 30,95440 Bayreuth, Germany

The determination of aldehydic compounds was achieved by gas chromatographic/mass spectrometric analysis of derived thioacetals. These were produced, together with artifacts, in a one-step reaction when plasmalogens were treated with ethane-1,2-dithiol and BF, . The artifacts were recognized to be 2-alkylidene-l,3dithiolanes derived from phospholipids. The latter were converted by BF, treatment into diacylglycerols, which were transformed in a complicated reaction into 2-alkyl-2- I (2-mercaptoethyl)thio] -1,3dithiolanes. These compounds were decomposed thermally, e.g. in the injector of a gas chromatograph, in Z-alkylidene-1,3-dithiolanes. This reaction is not restricted to phospholipids but occurs with all monoacylated glycols.

INTRODUCTION

Plasmalogens (1) are glycerides containing a long-chain aldehyde connected by an enol ether bond to the OH group in position 1 of glycerol in a neutral lipid (la) or a phospholipid (lb) (Scheme 1). Plasmalogens occur in all mammalian tissue and body fluids. They are espe- cially abundant in brain and heart, amounting up to 40% of the phospholipid fractions.'-5

CH2-O-CH:CH-RI

C;H.O-R,

CH,-0 -R,

I

- l a R, = alkyl l_b R, = alkyl R, = acyl R, = acyl R, = acyl R, = phosphocholine

phosphoethanolarnine

Scheme 1. Neutral and ionic plasmalogens 1.

We have already described a simple method to detect plasmalogen aldehydes and aldehydic lipid peroxidation products as 2-alkyl-1,3-dithiolanes (2).677 Besides 2- alkyl-1,3-dithiolanes (2), we could also recognize a homologous series of 2-alkylidene-1,3-dithiolanes (3) as artifacts. We consequently examined their origin and formation.

EXPERIMENTAL

Gas chromatography (GC) and gas chromatography/ mass spectrometry (GC/MS)

Measurements were carried out with a Carlo Erba HRGC 5160 Mega Series chromatograph equipped with a flame ionization detector, using a DB-1 fused- silica glass capillary column (30 m x 0.32 mm id., 0.1

* Dedicated to Prof. Dr. M a x Herberhold on the occasion of his

t Author to whom correspondence should be addressed. 60th birthday.

pm), temperature programmed from 80 to 280°C at 3 "C min-'. The temperature of the injector and detector were kept at 270 and 290°C, respectively. The carrier gas was hydrogen with a head pressure of 50 kPa and the splitting ratio was 1:30 (80%). Retention indices (RI) were determined according to Kovats* after addition of a mixture of straight-chain hydrocarbons (C10-C30) to each sample.

GC/MS was performed on a Finnigan MAT 95 system with an ICIS data system. Electron impact (EI) mass spectra were recorded at an ionization energy of 70 eV. An HP 5890 gas chromatograph with a DB-1 fused-silica column (30 m x 0.25 mm i.d., 0.25 pm) was used for sample separation. The carrier gas was hydrogen and the temperature programme was the same as used in GC.

'H NMR

Bruker AM 300, samples were dissolved in CDCl, .

Chemicals

Boron trifluoride ethyl etherate (48%), decanoic acid, 1,3-dipalmitoyl-glycerol, D,L-a$-distearin, ethane- 1,2-dithiol, KHCO, , methyl nonadecanoate, propane-1, 3-diol and p-toluenesulphonic acid monohydrate were purchased from Fluka (Neu-Ulm, Germany), 1,2-

dihexadecanoyl-sn-glycero-3-phosphate and trilaurin from Sigma (Deisenhofen, Germany), 1-undecenoic acid and silica gel PF,,, from Merck (Darmstadt, Germany), trimethyl orthovalerate from Aldrich (Steinheim, Germany) and MSTFA from Machery- Nagel (Diiren, Germany).

dihept adecanoyl-sn-glycero-3-phosphocholine, 1,2-

Formation of 2-octyl-2,3-dehydro-1,4-dithiaoe (6a)

2-(l-Hydroxy)nonyl-l,3-dithiolane (5a) was prepared as described Water elimination from 5a was effected by treatment of 0.1 mmol of 5s with 0.1 mmol

CCC 1076-5 174/96/030303-06 0 1996 by John Wiley & Sons, Ltd.

Received 14 September I995 Accepted 28 November 1995

304 A. DUDDA AND G. SPITELLER

of p-toluenesulphonic acid in 10 ml toluene for 1 h at 140 "C. After neutralization, the reaction mixture was extracted three times with diethyl ether and dried under reduced pressure. Compound 6a was isolated by thin- layer chromatography using cyclohexane-ethyl acetate (98 : 2, v/v) as mobile phase. The product was diluted 1 : 10 (w/v) in toluene and analysed by GC/MS and 'H NMR spectroscopy. 2-0ctyl-2,3-dehydro-1,4-dithiane (6a): R, = 0.44;

RI = 1801; MS (m/z; relative intensity, X) 230 (loo), 131 (79, 132 (70), 103 (35), 202 (15); 'H NMR, 6 5.82 (lH, CH, s), 3.07-3.23 (4H, SCH,CH, S , m), 2.12-2.18 (2H, CH,C, m), 1.17-1.27 (12H, (CH,),, m), 0.83-0.90 (3H, CH, , m).

Preparation of alkyl esters

1 rnol amount of acid and 0.75 mol of alcohol were dissolved in 200 ml chloroform and refluxed for 18 h using 3 ml of concentrated sulphuric acid as catalyst. The crude mixture was evaporated under reduced pressure and the residue was dissolved in cyclohexane and washed three times with 2 M KHCO, and water. The solvent was removed under reduced pressure. Separa- tion of mono- and diacyl esters was achieved by thin- layer chromatography with cyclohexane-ethyl acetate (9: 1) (20 x 20 cm plates, 0.75 mm PF254 silica gel 60). Detection was performed with 10% ethanolic H , [P(Mo,O ,J4] and heating.

For trimethylsilylation, 0.3 mg of each sample was dissolved in 10 p1 of ethyl acetate and 20 p1 MSTFA were added. The mixture was allowed to stand at room temperature for 12 h, then 0.5 1.11 was subjected to GC and GC/MS.

l-Decanoylethanediol (15a): R, = 0.08; RI = 1732 (TMS-ether); MS, m/z 73 (loo), 155 (85), 115 (63), 103 (40), 273 (20). 1,2-Didecanoyl-ethanethiol (16a): R, = 0.44; RI = 2469; MS, m/z 199 (loo), 155 (954, 217 (20), 243 (8), 370 (5). 2-Methoxyethyl-undec-10-enoate (17): R, = 0.55; RI = 1421; MS, m/z 41 (loo), 149 (80), 166 (77), 21 1 (lo), 242 (5). 3-Hydroxypropyl-undec-10-enoate (18): R, = 0.31; RI = 1918 (TMS-ether); MS, m/z 149 (loo), 73 (47), 299 (30), 224 (20), 314 (7). 3-Undec-10- enoyloxypropyl-undec-10-enoate (19) (1,3-diundec-l0'- enoylpropanediol): R, = 0.66; RI = 2783; MS, m/z 225 (loo), 113 (20), 149 (15), 167 (lo), 408 (5).

Preparation of S-propyl hexadecanetbiote (22)

0.1 rnol hexadecanoyl chloride and 0.1 mol of sodium propanethiolate were dissolved in 50 ml of methylene chloride and stirred for 2 h at room temperature, with exclusion of moisture. The preparation procedure was the same as described above.

S-Propylhexadecanthioate (22): RI = 2249; MS, m/z 239 (loo), 57 (50), 71 (35), 85 (25), 109 (lo), 271 (10).

Ditbiolane derivatization6

This was slightly modified using 0.1 mmol of each sample, 2 mmol of ethane-1,Zdithiol and 1 mmol of BF, in 5 ml of dry diethyl ether for 72 h, with exclusion

of moisture. The reaction mixtures were neutralized with 10% aqueous KHCO, and extracted three times with diethyl ether. The samples were dried under reduced pressure and analysed by GC and GC/MS after dilution with toluene 1 : 100 w/v).

2-Butylidene-1,3-dithiolane (3a): RI = 1295; MS, m/z 131 (loo), 160 (40), 132 (171, 71 (15). 2-Nonylidene-1,3- dithiolane (3b): RI = 1823; MS, m/z 131 (loo), 230 (35), 202 (20), 169 (15), 71 (13), 105 (8). 2-Pentadecylidene-l,3- dithiolane (3c): RI = 2469; MS, m/z 131 (loo), 253 (35), 314 (20), 286 (15), 105 (10). 2-Hexadecylidene-1,3- dithiolane (3a): RI = 2574; MS, m/z 131 (loo), 267 (43, 328 (35), 71 (18), 300 (15). 2-Heptadecylidene-l,3- dithiolane (3e): RI = 2687; MS, m/z 131 (loo), 281 (459, 342 (22), 314 (15), 105 (12).

Elemental analysis of 2-heptadecyl-2-[(2-mercaptoethyl)thio]-1,3-dithiolane (20a): expected, C 60.49, H 10.15, S 29.36; found, C 60.56, H 10.14, S 29.21%.

'H NMR (300 MHz, CDCl,): 6 (ppm), 2- heptadecylidene-1,3-dithiolane (3e), 5.49-5.5 1 (lH, CH=C, d), 3.27-3.38 (4H, SCH,CH,S, m), 2.05--2.12 (2H, CH,CH=, m), 1.13-1.28 [24H, (CH&, m], 0.82- 0.87 (3H, CH, , m); 2-heptadecyl-2-[(2-mercaptoethyl) thio]-1,3-dithiolane (20a), 3.27-3.50 (4H, SCH,-CH,S, m), 2.72-2.82 (4H, SCH,CH,SH, m), 2.13-2.19 (2H, CH,C, m), 1.57-1.67 (3H, CH,CH,C, S H , 33 = 8.0 Hz, tr), 1.15-1.34 [28H, (CH2)14, m], 0.82-0.87 (3H, CH,, m).

RESULTS AND DISCUSSION

The analysis of the distribution of aldehydes in plasmalogens is difficult. In most cases, tissue lipids were extracted according to Bligh and Dyer." The lipids were reduced with LiAlH, "L'~ to long-chain alcohols and separated from the glycerol enol ethers and other products by chromatography. The glycerol enol ethers were hydrolysed and the resulting aldehydes were reduced to alcohols. These were identified after acety- lation and separation by GC/MS.', Unfortunately, this procedure turned out not to be specific, since the separation of glycerol enol ethers from alcohols is often not ~omplete . '~

Therefore, we prefer to convert the enolic ethers directly into thioacetals, thus avoiding several steps of the above method. The crude lipid fraction containing neutral and ionic plasmalogens was treated with ethane- 1,2-dithiol in presence of BF, to produce 2-alkyl-1,3- dithiolanes (2).6*15-18 Af ter enrichment by thin-layer chromatography, the thioacetals were identified by GC/MS. This method offers the advantage of yielding derivatives of plasmalogen aldehydes directly and also of aldehydic compounds derived by lipid peroxidation of unsaturated fatty acid substituents.'

Most spectra of 2-alkyl-1,3-dithiolanes (2) are characterized by a base peak at m/z 105, the molecular ion and a [M - 611' fragment (Fig. 1).

In addition to these thioacetals, we detected a homologous series of 2-alkylidene-1,3-dithiolanes (3), which were characterized in their mass spectra by a base peak at m/z 131, molecular ions and further key fragments such as [M - 28]+ and [M - 611' (Fig. 2).

100-

10.

60

40

20

283 I ~1 GI 154 ias 227 250 2 a q

STRUCTURE AND ORIGIN OF ARTIFACTS IN THE ANALYSIS OF PLASMALOGENS

I 1

I

10-

60-

40-

20 -

I E+ 07

so 1 F

,s - CH,

MW 344

150 200 2 so 300 350 400

2.33

305

Figure 1. Mass spectrum of 2-heptadecyl-1.3-dithiolane (2a).

Peak matching revealed the elemental composition of the ion at m/z 131 to be C,H,S,. Thus structure 4 was proposed for it, and structure 3 was derived for the original compound (Scheme 2). Synthesis of 3 was attempted via elimination of water from corresponding a-hydroxyaldehyde 5, but the RI, mass and NMR spectra of the synthetic product and that derived from natural sources were not identical (Scheme 3 and Fig. 3).

The NMR spectrum of the dehydration product showed a singlet at 5.82 ppm typical of a ,C=CH-S- structural element, hence structure 6a \

was ascribed to the dehydration product of 5a. The elimination of water had obviously occurred via the intermediates 7a and 8a, leading to 6a. Therefore, 2- alkylidene- 1,3-dithiolanes 3 could not have been derived from a-hydroxyaldehydes 5, i.e. the 2-alkylidene-1,3- dithiolanes 3 must have other precursor molecules. Consequently, we investigated the behaviour of different tissue components by treatment with ethane-1,Zdithiol and BF,. If we treated phospholipids 9 or 10 [1,2- diheptadecanoyl-sn-glycero-3-phosphocholine (9a), 1,2- dihexadecanoyl-sn-glycero-3-phosphate (10a)l with

I 100 3 131

I ,s - CH,

MW 342

281 I

34a

I

50 100 150 200 250 300 350 400

Figure 2. Mass spectrum of 2-heptadecylidene-l,3-dithiolane (39)


Scheme 2. Formation of the ion at m/z 131 from 2-alkylidene-1 ,a-dithiolanes (3) by El MS.

these reagents, we detected 2-alkylidene-l,3-dithiolanes 3 in the reaction mixture. Further investigation revealed that 2-alkylidene-1,3-dithiolanes 3 are produced also by 1,2-diacylglycerols 11 and 1,3-diacyglycerols 12 [ 1,2-distearoylglycerol (lla), 1,3-dipalmitoylglycerol (12a)], but not by triacylglycerols 13 [trilauroylglycerol (13a)l or simple methyl esters 14 [methyl nonadecanoate (14a)l. They are also produced by monoacylated glycols 15 [l- decanoylethanediol(15a)], but not by diacylated glycols 16 [ 1,2-didecanoylglycol (Ma), 2-methoxyethyl-undec- 10-enoate (17), 3-hydroxypropyl-undec-l O-enoate (18) and 1,3-diundec-lO-enoylpropanediol (19)]. Thus the minimal structural requirements to produce the 2-

alkylidene-1,3-dithiolanes 3 seem to be of monoacylated glycol moieties (see Scheme 4).

Elemental analysis and 'H NMR spectroscopy of the reaction product of 1,2-distearoylglycerol (lla) with ethane-1,2-dithiol and BF, revealed 2-heptadecyl-2-[(2- mercaptoethyl)thio]-1,3-dithiolane (20a) as a product instead of the expected 2-heptadecylidene-l,3-dithiolane 3e (Scheme 5).

When 20s was introduced into the GC/MS system it decomposed quantitatively by loss of ethane-1,Zdithiol to heptadecylidene- 1,3-dithiolane (3e).

Considering the reaction mechanism, S-(2-mercap- toethy1)alkyl esters 21 may be intermediates in the reac-

3b

Scheme 3. Formation of 2-octyl-2.3-dehydro-I.4-dithiane (6a) by elimination of water from 2-(1 -hydroxy)-nonyl-l,3-dithiolane (58).

105 40-

I

20-

so 100

I

7.34

M W 230

Figure 3. Mass spectrum of 2-octyl-2.3-dehydro-I.4-dithiane (Ba).

STRUCTURE AND ORIGIN OF ARTIFACTS IN THE ANALYSIS OF PLASMALOGENS 307

$H2.0COR :HZ' OCOR CH-OCOR R=Cl& S;H,-OCOR

I L9 R=C,oH,, CH,- phosphocholine S;H-OCOR R=C&,j CH,-OCOR CH2-OPOjHz

YHz* OCOR

CH,-OH / S;H-OCOR 2

/ t i ?-I2* OCOR

R=C,oH19 w cH,- cH= c / \ '3 R=C17Hs

YH,-OCOR CH,-OCHj

- 18 CH,

~H,-oH 1

\I YH-OH YHz*oCoR 12a

/ 3 S base peak rn/z=131

CH,. OCOR + 5H2'0C0R R=C15H31

R=C18HJ7 CH2- OCOR

/ i R=C,oH19 YH,-OCOR CH,.OCOR YH,.OCOR R-COOCH, YH-OCOR

R=C&g CHZ-OH 13a - 16a R=CgH,g - 14a R=C1

15a R = R' -CH,-CHz- -

Scheme 4. Precursor molecules of P-alkylidene-l,3-dithiolanes 3 by treatment with ethane-l,2-dithiol and BF, .

1 SH

OTHEG

Scheme 5. Formation of 2- heptadecyl-2-[(2-mercaptoethyl)- thiol-I ,3-dithiolane (20a) from 1.2-distearoylglycerol (1 1 a) fol- lowed by thermal decomposition (GC conditions) providing 2- heptadecylidene-I ,3-dithiolane (38).

tion. Thus, S-propyl hexadecane thioate (22) was prepared and treated with ethane-1,2-dithiol and BF, . Unfortunately, pentadecylidene-1,3-dithiolane (3d) was found in a yield of only 7%.

Alternatively, 2-alkyl-2-[(2-mercaptoethyl)thio]-l,3- dithiolanes (20) could also be obtained from an activat- ed internal orthoester which is attacked by ethane-1,2- dithiol. The last step of this reaction is the cleavage of the ester bond. Therefore, trimethyl orthovalerate (23) was treated with ethane-1,3-dithiol and BF, . 2- Butylidene-1,3-dithiolane (3a) could be detected in a yield of 70% in addition to a dimerization product. 2-Alkyl-2-[(2-mercaptoethyl)thio]-1,3-dithiolanes (20)

can be obtained in nearly 100% yield from the corresponding monoglycol ester derivatives 15. Their thermal decomposition products represent stable derivatives of the corresponding ketenes and may therefore also be useful starting materials for synthesis.

Acknowledgements

We thank the Deutschen Forschungsgemeinsch~t and the Fonds der Chemischen Industrie for financial support. We are obliged to Mr M. Glaessner for recording the mass spectra. We are also grateful to Mr W. Kern for purification of solvents.

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structure and origin of artifacts in the analysis of plasmalogens

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