The ALK-i -enyl ether and alkyl ether lipids of bovine heart muscle

BIOCHIMICA

ET BIOPHYSICA

141

ACTA

BBA 55498

THE ALK-I-ENYL

ETHER

HEART

MUSCLE

HARALD

H. 0. SCHMID

ofM~~neso~~, (Received Pilaygth, I#%) ~~~v~rs~~y

AND

AND ALKYL

TAKASHI

The Ha~~ae~.~~t~~~e*

ETHER

LIPIDS

OF BOVINE

TAKAHASHI A ustin,

M&m.

55912

f U.S.A .)

SUMMARY

Choline phosphatides and ethanolamine phosphatides as well as neutral afkoxyhpids and triglycerides were isolated from a lipid extract of bovine heart muscle. The constituent alk-I-enyl glycerol ethers, alkyl glycerol ethers and fatty acids of these lipid classes were analyzed after selective acidic or enzymatic hydrolysis. Comparative analyses of the aliphatic moieties at the I and z positions of the phosphatides revealed a pronounced similarity of the three classes of choline phosphatides, whereas significant differences between the various ethanolamine phosphatides were encountered. Alk-I-enyl diglycerides and alkyl diglycerides differed in the composition of their aliphatic moieties from each other, from the triglycerides and from the phosphatides.

INTRODUCTION

Alk-I-enyl ethers and alkyl ethers of glycerol occur as constituents of phosphatides and of neutral lipids. Whereas alk-r-enyl acyl phosphatides (plasmalogens) have been studied extensively’, the isolation and analysis of alk-x-enyl diglycerides (neutral plasmalogens) has been achieved only recentlyaTg. The plasmalogens of bovine heart muscle are choline phosphates and ethanolamine phosphates of O-&S-Ialk-I’-enyl-a-acyl-glycerol. Neutral plasmalogens and alkyl diglycerides have been detectedGy in extracts of beef heart and have been isolateda. The presence of small amounts of alkyl acyl phosphatides has also been demonstrated’. Analyses of aldehydes and fatty acids released from the plasmalogens of bovine heart muscle have been reporteds~*, but comparative studies of all aliphatic moieties of the various ionic and neutral alkoxylipids have not been performed. A review10 on the biochemistry of lipids containing ether bonds has appeared recently. The biosynthesis of the alk-I-enyl ethers and alkyl ethers of glycerol is not yet fully understood and a metabolic relationship between alkoxylipids and the corresponding ester lipids has not been conclusively demonstrated. However, as a direct metabolic conversion of diacyl phosphatides or diglycerides to the corresponding alkB&whim.

Biopkys.

X&%X, 164 (1968)

141-147

I42

H. 0. SCHMID,

T. TAKAHASHI

I-enyl acyl and/or alkyl acyl derivatives appears to bepossiblell, the aliphatic moieties of the presumable metabolites are of special interest. This communication reports analyses of the constituent alk-I-enyl ethers, alkyl ethers and fatty acids of the I and 2 positions of choline phosphatides, ethanolamine phosphatides, and of the neutral alkoxylipids and triglycerides of bovine heart muscle. MATERIALS

Lipid

AND

METHODS

extract

Three beef hearts were obtained immediately after slaughter. The muscle tissue was cut and homogenized in a Servall Omnimixer with chloroform-methanol (2 :I, v/v). The total lipids were extracted and purified 12, dried in vaczho,weighed and redissolved in chloroform. From 2.9 kg of bovine heart muscle, 67.5 g of lipid material was obtained. Isolation

of lipid

classes

Lipid classes, both neutral lipids and phosphatides, were tentatively identified by thin-layer chromatography 13. Solutions of ninhydrin and z,4_dinitrophenyl hydrazine, molybdenum blue reagent, Dragendorff’s reagent and chromic sulfuric acid were used as indicators. Neutral lipids were fractionated as previously describede. A concentrate of alk-I-enyl diglycerides and alkyl diglycerides was applied to a thin-layer chromatography plate and exposed to HCI fumes. The resulting aldehydes and diglycerides were then separated273 from each other and from the unchanged alkyl diglycerides by thin-layer chromatography. Triglycerides were isolated from the total lipids by thin-layer chromatography. Choline phosphatides and ethanolamine phosphatides were separated on layers of silica gel H (E. Merck, Darmstadt, Germany), 0.5 mm thick, on glass plates 20 cm x 20 cm. Approx. 60 mg of lipid was applied per plate and chloroform-methanol-water (65 :25:4, by ~01.)~~was used as the developing solvent. Fractions were detected by spraying the plates with water; scraped off, eluted15, and rechromatographed, 30 mg per plate, with chloroform-methanol-acetic acid-water (25 : 15 : 4: 2, by ~01.)~~. Ry combining corresponding fractions and refractionation, pure choline phosphatides and ethanolamine phosphatides were obtained without preferential loss. The purity of the lipid classes isolated was ascertained by two-dimensional thin-layer chromatography” and by analyzing the products of chemical and enzymatic reactions. Chemical

reactions

Experimental conditions of reactions leading to derivatives suitable for analysis of the aliphatic moieties have been described previously293: aldehydes were reduced to alcohols with LiAlH,, alcohols were acetylated with acetic anhydride; methyl esters were prepared through methanolysis of the respective lipids with methanolHCl; alkyl glycerol ethers were obtained by reduction of the phosphatides with lithium aluminium hydride followed by thin-layer chromatography, isopropylidene derivatives of alkyl glycerol ethers were prepared in acetone containing catalytic amounts Biochim. Biophys. Acta. 164 (1968). 141-147

ALK-I-ENYL

I43

ETHER AND ALKYL ETHER LIPIDS

of perchloric acidIs. Methyl esters and alkyl acetates were hydrogenated in ethyl acetate using PtO, as a catalyst. Alk-r-enyl acyl choline phosphatides and ethanolamine phosphatides were hydrolyzed with acetic acid; portions (goo mg) of both the total choline phosphatides and ethanolamine phosphatides were dried in r’acuo and incubated with IO ml of 90% acetic acid for 18 h at 38” to yield aldehydes and (z-a+) lysophosphatidess. The lysophosphatides obtained were separated from the unchanged diacyl phosphatides and alkyl acyl phosphatides by thin-layer chromatography (30 mg of lipid per plate, chIorofo~-methanol-water (65 : 25 : 4, by vol.)). Aldehydes were isolated from the reaction mixture by thin-layer chromatography (IOO mg per plate, hexanediethyl ether (90: IO, v/v)).

Diacyl choline phosphatides and ethanolamine phosphatides free of plasmalogens but containing small amounts of alkyl acyl phosphatides, were incubated with phospholipase C (EC 3.1.4.3) 1sg20 in Tris buffer. Choline phosphatides (200 mg) were shaken in a zoo-ml flask with roo ml buffer containing IO mg phospholipase C*, 50 ml diethyl ether were added and the mixture was stirred vigorously at room temperature. Samples were checked every z h by thin-layer chromatography (chloroform-methanol-water (65 :25 :4, by vol.) ; hexane-diethyl ether (50:50, v/v)). Hydrolysis was complete after 12 h. The reaction mixture was extracted 3 times with hexane; the hexane solution was washed with water, dried over Na,SO,, evaporated under reduced pressure and reacted with acetic anhydride in pyridine at 80” for 2 h. Ethanolamine phosphatides (zoo mg) were not emulsified with Tris buffer and products of hydrolysis were not observed after incubation with phospholipas~ C for 18 h. However, the addition of an equal amount (2-acyl) choline lysophosphatides derived from the corresponding plasmalogens effected complete hydrolysis within 18 h (ref. 21). Hydrolysis with phospholifiase A Diacyl choline phosphatides and ethanolamine phosphatides were hydrolyzed with phospholipase A (EC 3.r.r.4fz2. The phosphatides (20 mg) were dissolved in 5 ml diethyl ether and 0.5 ml Tris buffer containing CaCI,andphospholipaseA** (5 mg,/ml) were added. Hydrolysis of the choline phosphatides was complete after 3 h. The ether layer was removed and the aqueous phase was washed 3 times with diethyl ether. The free fatty acids in the ether were checked for purity by thin-layer chromatography and converted to their methyl esters with methanol-HCI. The aqueous phase was brought to dryness under reduced pressure and the (I-acyl)choIine lysophosphatides were redissolved in a small amount of chloroform-methanol (z:I, v/v) and checked by thin-layer chromatography; they were then subjected to methanolvsis. Hydrolysis of the ethanolamine phosphatides was complete after 24 h;. the ether was removed and the aqueous phase washed as described above. However, some (I-acyl)ethanolamine lysophosphatides were detected in the ether phase and * Lecithinase-C from Closfridium welchii, Sigma Chemical Co., St. Louis, MO.. U.S.A. ** Lyophilized venom of Crotalus adamanteus, Ross Allen’s Reptile Institute, Inc., Silver Springs, Fla., U.S.A. Biochim. Biophys.

Acta, 164 (1968) rqr-147

I44

H. 0. SCHMID, T. TAKAHASHI

were separated from the fatty acids by thin-layer chromatography (chloroformmethanol-water, 70 : 30 : 3, by vol.) The fatty acids and (I-acyl)lysophosphatides, combined with the residue of the aqueous phase, were reacted with methanol-HCl without Analysis

prior elution from the adsorbent. of aliphatic

constituents

Methyl esters, alkyl acetates,

aldehydes

and isopropylidene

derivatives

of alkyl

glycerol ethers were analyzed by gas chromatography. Peaks were assigned by comparison with standards and through their relative retention times23. Prefractionation of complex mixtures by argentation thin-layer chromatographyz4, aided in assigning fractions methyl

in the gas chromatograms. esters and alkyl acetates

Gas chromatography A Beckman instrument,

Gas chromatographic

were performed

chain length

after catalytic

Model GC-zA, equipped

analyses

of

hydrogenation.

with a flame ionization

detec-

tor and an aluminium column, zoo cm in length and 4 mm inner diameter, packed with 20 “/(, ethylene glycol succinate and 2 o/o phosphoric acid on 80-100 mesh Gas Chrom P, was operated of peaks. Standard

at 180’ and 200’. Quantitative

mixtures

of methyl

esters were purchased

Lipids Preparation Laboratory, aldehydes 25, derivatives alkyl acetates2 were prepared as described previously. RESULTS

analysis

was by triangulation

from The Hormel Institute of alkyl glycerol ethersze and

AND DISCUSSION

Choline phosphatides were isolated.

After

and ethanolamine

mild acidic hydrolysis

phosphatides

of bovine

of the plasmalogens

heart

muscle

the aldehydes

and

(z-acyl)lysophosphatides as well as the unchanged diacyl phosphatides including small amounts of alkyl acyl phosphatides were isolated. The aldehydes and the constituent fatty acids of the (2-acyl)lysophosphatides were analyzed by gas chromatography. We observed that part of the aldehydes formed during hydrolysis underwent condensation to z,3-dialkyl acroleinsZs. However, this reaction did not result in any preferential losses of aldehydes and did not influence their analyses. Reduction of the “diacyl” phosphatides with LiAlH, and subsequent thin layer chromatography revealed small amounts of glycerol ethers indicating the presence of alkyl acyl phosphatides. Therefore, the choline phosphatides and ethanolamine phosphatides were hydrolyzed with phospholipase C, and the diacyl glycerols and alkyl acyl glycerols produced were acetylated. The amount of the alkyl acyl glycerol acetates was estimated to be about 5 “/Aof the diacyl glycerol acetates for those derived from the choline phosphatides and 39/o for those derived from the ethanolamine phosphatides. The acetates were isolated and methanolysis of the alkyl acyl glycerol acetates produced glycerol ethers and methyl esters for gas chromatographic analysis. The amounts of alkyl acyl phosphatides relative to the diacyl phosphatides were considered negligible for the analysis of the constituent fatty acids of the latter. Thus, the diacyl phosphatides were hydrolyzed with phospholipase A. The fatty acids obtained through hydrolysis and the constituent fatty acids of the (I-acyl)lysophosphatides were analyzed. The results of the analyses of all aliphatic moieties at Biochim.

Biophys.

Acta,

164 (1968)

141-1.$7

ALK-I-ENYL

ETHER AND ALKYL ETHER LIPIDS

145

I

TABLE ALIPHATIC

MOIETIES

IN THE

I POSITION

OF CHOLINE

PHOSPHATIDES

AND

ETHANOLAMINE

PHOSPHATIDES

br, total “branched

chain” compounds

(after hydrogenation)

Choline phosphatides

Chain length and number of double bonds

. Fatty

I4 br* 14:o I5 br* 15 br**

tr tr tr tr

15:o

17 br* 17 br** 1710

2.6

3.9 1.8

13.9 8.0

18:o 18:1

18:~ 20 (total)

tr

0.5 4.5 1.0

I.4 62.0 I.9 3.3 4.7 2.5 II.0 7.0

2.7

16:1

0.5 3.5

0.5 0.7 0.6 1.6 2.8

0.5 tr 62.3

br* 16:o

16

ALkyl ethers***

Alk-I-enyl ethers**

acid>*

51.8 tr 3.4 5.1 I.2 10.1

18.4 -

tr ?

3.2

tr

1.1

;

tr, traces, less than 0.4.

Ethanolamine

tr

phosphatides Alk-I-enyl ethers**

Fattv acids* tr tr tr tr tr tr

Alkyl ethers***

tr

tr

0.4

0.6 tr tr

tr

3.0

tr tr tr tr 88.1 6.0

0.4 I.0 0.6 28.8 I.I I.3 2.4 3.2 42.7

2.4 42.1

18.1

18.7

tr

tr

I.3 1.6

0.9

tr

30.5 tr 2.8 2.0

* As methyl esters. ** As alkyl acetates. *** As isopropylidene derivatives.

TABLE

II

CONSTITUENT FATTY ACIDS OF THE 2 POSITION OF CHOLINE PHOSPHATIDES AND

ETHANOLAMINE

PHOSPHATIDES

tr, traces, less than 0.4.

Chain length, number and position* of double bonds

Choline phosphatides

16:0**

4.8 3.1 0.4 28.9 45.5 0.8 5.1

Diacyl

16:1 18:0 18:1

18:~ 9, 12, 15-18:3 8, II, 14-20:3 5, 8, II, 14-20:4 5. 8, II, 14, 17-20:5 7, IO, 13. 16-22:4 7, IO, 13, 16, 19-22:5 * Major isomer. ** Traces of I4:0, listed.

the I and Tables

2 positions

10.6

0.7 0.7

I.3 I.7 13.1

12.0

53.0 0.6

10.4 0.4 0.6 tr

0.5 1.7 I.4

50.7 0.5 5.3 14.2 tr 1.6 1.0

Diacyl

phosphatides

Alk-I-enyl acyl

0.5

A Lkyl acyl

I.5

tr

tr

2.7 3.2 13.0

4.4 3.7 34.3

2.4 73.0 3.0 I.0 1.2

6.6 42.2 1.3 2.9 3.1

tr

tr

5.6 I.1

5.6 8.8 17.6 0.9 6.0 27.3 5.9 4.4 16.8

19:0, 20:0, 20: I and 22: 6 were also present in all lipid classes

of the choline

and ethanolamine

phosphatides

are listed

in

a pronounced

similarity

in

I and II. A comparison

the composition phosphatides. tuents;

Alkyl acvl

2.2

6.1 21.1

15:0, 17:o.

Ethanolamine

Alk-I-envl acyl

of the data listed in Table

of all aliphatic

I reveals

moieties in the I position

In these classes the 16:o

among the minor constituents

of the three types of choline

and 18: o derivatives

are the major

are two series of “branched Biochim. Biophys.

consti-

chain” compounds.

Acta, 164 (1968)

141-147

H. 0. SCHMID, T. TAKAHASHI

146

In contrast, the composition of the constituent alk-I-enyl ethers and alkyl ethers of the ethanolamine phosphatides is the same, but it is strikingly different from that of the corresponding fatty acids. In particular, there occurs very little palmitic acid whereas the corresponding hexadec-I-enyl ether or hexadecyl ether are major components of the ethanolamine phosphatides, a fact noted also for other tissues29. Our findings indicate that the major portion of the diacyl ethanolamine phosphatides cannot be a direct precursor of any of the alkox~lipids, whereas the structural similarity among the choline phosphatides supports the view of a direct metabolic relationship between them. The resemblance in the composition of the fatty acids in the z position of the three classes of choline phosphatides (Table II) is less pronounced, but also less significant than that of the aliphatic moieties in the I position. Table III gives the results of analyses of the aliphatic moieties in neutral alkoxylipids and triglycerides. Total aliphatic moieties of the ~kox~lipids were calculated from the percentages given for ethers and fatty acids, whereby the alkoxy moieties contribute one-third and the acyl moieties two-thirds of the total percentage. The alk-I-enyl ether composition of the alk-r-enyl diglycerides is characterized by a large amount of a substance resembling “branched pentadecanal” in the aldehydes obtained by hydrolysis. As the same amounts of a “branched &,-compound” were found in free aldehydes as well as in alkyl acetates derived thereof, the correTABLE

III

ALIPHATIC

MOIETIES

OF ALK-I-ENYL

DIGLYCERIDES,

ALKYL

DIGLYCERIDES

AND

TRIGLYCERIDES

br, total “branched chain” compounds (after hydrogenation); tr, traces, less than 0.4. Fatty acids as methyl esters: alkyl ethers as isopropylidene derivatives; total aliphatic moieties are calculated by considering alk-renyl ethers or aIky1 ethers as I/$ and fatty acids as s/3 and adding the corresponding percentages. -_ --. .__ AEk-r-PW$ diglycevides Trigl_ycerides Ckaiz leagth, nwnzbev A Ekyl di&cerides and positim* of Total Fatty Total Alk-x-my1 Fatty A Ekyl Fatty doxble bokzds acids ethers acids ethers acids 14 br* r4 br** 14:* 15 hr* r5 br**

2.2 -

tr

‘2.0

I.1

1.8 9.3

1fJ:0

5.0

2.4 0.8 0.4 13.7

38.1 3.8 0.7 4.5 1.6 23.9

rh:o 16:1

17 br* r7 br** 17:o IS br* x8:0 18:1 18:~

-7.1

9. 5, 5, 7. 7,

-

12, Is-18:3 *I, 14-20:3 8, II, 14-20:x$ 10, 13. 16-22:4 IO, 13, 16, 1g-22:5

* ** *** t

2.0

0.7 tr I,4

tr 4.0

-

2.2

tr

2.3

3.7

1.5 2.7 32.3 tr

I.0 tr

2.0 21.9 2.6

0.4 0.5 tr tr 15.5

0.5 I.8 0.6 tr 18.4

27.0 21.8

21.0 -

-

-

2.6 6.1 2.3

20.4 14.6 0.6 x.7 4.1 I.5

-

2.5

1.7

-

0.9

Major isomer. As alkyl acetates. Total C,,, 1.7. Total C,,, 0.7. Rioclzim. Bioph_vs. dcta,

16.1 (1968) 141-147

1.0

3.4

22.6

2.3

tr 0.7 3,5 I.6 I.” 0.9 25.6 1.5

I.1

I.2

I.2

2.4 ‘.?I 33.5

0.9 I.4 tr

I.4 I.4 tr

-

1.8 tr

tr 0.5 19.3 2.0 tr tr 2.0

1.0

21.3

25.2

18.5

29.0 4.4 0.9 tr 0.5 3.3 4-5

26.4 3.0 0.6 tr 0.3 2.2 3.0

4::: 0.9 tr*** tr trt tr

ALK-I-ENYL

ETHER AND ALKYL

ETHER LIPIDS

147

sponding alk-I-enyl ether appears to be a genuine constituent of the neutral plasmalogens of bovine heart muscle. It is interesting to note that large amounts of this compound were also detected in the phosphatides of ox spleena. The alkyl ether composition of the alkyl diglycerides was found to be similar to that of the alkyl acyl-ethanolamine phosphatides, but the alkyl diglycerides were almost free of arachidonic acid. It is a striking fact that the composition of the aliphatic moieties of this lipid class resembles that of alkyl diglycerides isolated from other tissues such as human periphric fat2 and subcutaneous tissue30 more closely than any of the alkyl acyl phosphatides of the same organ. ACKNOWLEDGEI\fENTS

This investigation was supported in part by Public Health Service research grants No. CA 10155 and HE 08214 and by The Hormel Foundation. The authors thank Dr. H. SCHLENKand Mr. T. GERSON,of The Hormel Institute, for a preparation of phospholipase A. REFERENCES I M. M. RAPPORT AND W. T. NORTON, Aszn. Rev. B&hem., 31 (1962) 103. 2 H. H. 0. SCHMID AND H. K. MANGOLD, Biochem. Z., 346 (1966) 13. 3 H. H. 0. SCHMID, W. J. BAUMANN AND H. K. MANGOLD, Biochim. Biophys. Acta, 144 (1967) 344. 4 J. C. M. SCHOGT, P. HAVERKAMP BEGEMANN AND J. KOSTER, J. Lipid Res., I (1960) 446. 5 J. R. GILBERTSON AND M. L. KARNOVSKY, J. Biol. Chem., 238 (1963) 893. 6 H. H. 0. SCHMID, L. L. JONES AND H. K. MANGOLD, J. Lipid Res., 8 (1967) 692. 7 R. PIETRUSZKO AND G. M. GRAY, Biochim. Biophys. Acta, 56 (1962) 232. 8 E. KLENK AND G. KRICKAU, Z. Physiol. Chem., 308 (1957) gS. g G. 111.GRAY, Biochem. J., 77 (1960) 82. IO F. SNYDER, in R. T. HOLMAN, Advances in the Chemistry of Fats and Other Lipids, Vol. IO, Pergamon Press, New York, 1968, in the press. II R. BICKERSTAFFE AND J. F. MEAD, Biochemistry, 6 (1967) 655. 12 J. FOLCH, M. LEES AND G. H. SLOANE-STANLY, J. Biol. Chem., 226 (1937) 497. 13 H. K. MANGOLD, in E. STAHL, Diinnschicht-Chro~~atographie, Edition 2, Springer Verlag, Berlin, 1967, p. 350. 14 H. WAGNER, L. HGRHAMMER AND P. WOLFF, Biochem. Z., 334 (1961) 175. 15 K. H. SLOTTA, Monatsh. Chem., g7 (1966) 1723. 16 V. P. SKIPSKI, R. F. PETERSON, J. SANDERS AND M. BARCLAY, J. Lipid Res., 4 (1963) 227. 17 J, G. PARSONS AND S. PATTON, J. Lipid Res., 8 (1967) 696. 18 D. J. HANAHAN, J. EKHOLM AND C. M. JACKSON, Biochemistry, z (1963) 630. 19 M. G. MACFARLANE AND B. C. J. G. KNIGHT, B&hem. J., 35 (1941) 884. 20 0. RENKONEN, J. Am. Oil Chemists’ Sot., 42 (1965) 298. 21 T. TAKAHASHI AND H. H. 0. SCHILIID, Chem. Phys. Lipids, in the press. 22 C. LONG AND I. F. PENNY, Biochem. J., 65 (1957) 382. 23 H. H. HOFSTETTER, N. SEN AND R. T. HOLMAN, J. Am. Oil Chemists’ Sot., 42 (1965) 537. 24 L. J. MORRIS, J. Lipid Res., 7 (1966) 717. 25 H. K. MANGOLD, J. Org. Chem., 24 (1959) 405. 26 W. J. BAUMANN AND H. K. MANGOLD, J. Org. Chem., 29 (1964) 3055. 27 W. J. BAUMANN AND H. K. MANGOLD, Biochim. BiophTs. Acta, 116 (1966) 570. 28 H. H. 0. SCHMID AND T. TAKAHASHI, Z. Physiol. Chem., in the press. zg G. M. GRAY AND M. G. MACFARLANE, B&hem. J., 81 (1961) 480. 30 H. H. 0. SCHMID, N. TUNA AND H. K. MANGOLD, Z. Physiol. Chem., 348 (1967) 730,

B&him.

Biophys. Acfa, 164 (1968) 141-147