542

M E T H O D S i

Stereospecific Analysis of Triacyl-sn-glycerols by Chiral High-Performance Liquid Chromatography Tom Takagi" and Yasuhiro Ando Department of Chemistry, Faculty of Fisheries, Hokkaido University, Minoto-cho, Hakodate, Japan 041

A method for the stereospeeific analysis of triaeyl-sn- glycerols by hlgh-performance liquid chromatography (HPLC) on a chiral column is described. "13riaeyl-sn- glycerols were partially degraded with ethyl magnesium bromide, and the monoaeylglycerols produced were sepa- rated as 1- and 2-monoaeylglycerol fractions by thin-layer chromatography on boric add-impregnated silica gel plates. The 1-monoacylglycerols were resolved into sn-1 and sn-3 monoaeylglyeerol fractions by HPLC on a chiral column (Snmlchiral OA-4100) after derivatization with 3,5-dlnltrophenyl isocyanate. Fatty acid methyl esters ob- tained from the original triacyl-sn-glycerols, 1- and 2- monoaeylglycerol fractions, and sn-1 and sn-3 monoaeyl- glycerol fractions were analyzed by gas chromatography on open-tubular eolnmnR. Stereospecific aeyl distributions in triacyl-sn-glyeerols were calculated from the data. The aeyl distributions of several oils were obtained. The method is rapid, simple and gave reproducible results. Lipids 26, 542-547 (1991).

The first stereospecific analysis of triacyl-sn-glycerols was reported by Brockerhoff in 1965 (1). The method involved the stereospeciflc cleavage with phospholipase A2 to distinguish between the sn-1 and sn-3 positions in a triacyl-sn-glycerol molecul~ Later, Lands and co-workers (2), Brockerhoff (3), Myher and Kuksis (4) and Christie and Moore (5) presented improved procedures for the stereo- specific analysis of triacyl-sn-glycerols. The methods were enzymatic and involved the stereospeciflc lipolysis or esterification of diacylglycerols or their derivatives to discriminate between the chiralities (6,7).

The present paper presents a novel method for the stereospecific analysis of acyl distributions in triacyl-sn- glycerols. In this analytical approach, high-performanee liquid chromatography (HPLC) of monoacylglycerol enan- tiomers on a chiral stationary phase is employed instead of stereospecific enzyme reactions. We previously intro- duced part of the methodology (8). Triacyl-sn:glycerols were partially hydrolyzed by ethyl magnesium bromide, and the 1-monoacylglycerol products were resolved into sn-1 and sn-3 enantiomers by HPLC as 3,5-dinitrophenyl- urethanes (3,5-DNPU) on a chiral column (Sumipax OA-2100, S-mitemo Chemical Ca, Osaka, Japan). Stereo- specific distributions of acyl groups were calculated from area percentages of the enantiomer peak. On the other hand, Laakso and Christie (9) reported the chromato- graphic resolution of diastereomeric 1,2-diacylglycerol derivatives prepared with a chiral reagent on a column of silica gel for the stereospecific analysis of triacyl-sn- glycerols. Fatty acid residues of the sn-l,2- and the sn-2,3-diacylglycerol derivatives were converted to methyl esters, and then analyzed by gas chromatography (GC).

*To whom correspondence should be addressed. Abbreviations: 3,5-DNPU, 3,5-dinitropbenylurethane; GC, gas chromatography; HPLC, high-performance liquid chromatography; TLC, thin-layer chromatography.

The acyl compositions at sn-1, sn-2, and sn-3 positions were then calculated.

We recently improved the resolution of the monoacyl- glycerol enantiomers by modification of the HPLC method {101. The method permits the clear separations of a 1-monoacylglycerol mixture into sn-1 and sn-3 mono- acylglycerols. In the present study, this modified HPLC method was applied to the stereospecific analysis of triacyl-sn-glycerols. The 1-monoacylglycerol products from the partial hydrolysis of natural triacyl-sn-glycerols were resolved into sn-1 and sn-3 monoacylglycerol fractions by a modified HPLC method, and the methyl esters prepared from the sn-1 and sn-3 fractions were analyzed by GC. The distribution of acyl groups between the sn-1, sn-2 and sn-3 positions was calculated based on the results of GC analyses.

MATERIALS AND METHODS Td~/l-sn-glycerols. Yew (Tzxus cusp id) seeds were col- lected at Hakodate, Japan. H igh erucic acid rapeseeds were obtained from Yonezawa Seiyu Ca (Saitama-ken, Japan}. Hawaiian macadarnia nuts and soybean oil were purchased at Hakodate. Rapeseed and macadamia nut oils were prepared from seeds or nuts by crushing and diethyl ether extraction. Yew seeds and flesh meats of herrings were extracted by the method of Bligh and Dyer {11}. Triacyl-sn-glycerol samples were purified by TLC on Kiesel Gel 60G plates {Merck, Darmstadt, Germany) with n- hexane/diethyl ether (4:1, v/v) for development. 1,3-Di- pMmitoyl-2-1inolenoylglycerol was prepared from 1,3- dipMmitoylglycerol by esterification of the secondary hydroxy group with linolenoyl chloride 1,3-Dioleoyl-2- stearoylglycerol was obtained from Sigma Chemical Co. (St. Louis, MO).

Partial hydrolysis of triacyl-sn-glycerols. The procedures for the analysis of triacyl-sn-glycerols are outlined in Scheme 1. The first step is the partial hydrolysis of tri- acylglycerol with ethyl magnesium bromide by a modi- fied Brockerhoff procedures (5,6). Triacyl-sn-glycerols (100 rag) were dissolved in 3 ml, of dry diethyl ether, and ethyl magnesium bromide in dry diethyl ether (0.33 mL of 3 M solution) was added. The mixture was shaken for 1 min, and then 0.1 mL glacial acetic acid followed by

1 chiral r ~ - EtMgBr TLC HPLC as

i SCHEME 1. Procedures for stereospecific analysis of acyl distribu- tion in triaeyl-sn-glycerols by chiral HPLC.

LIPIDS, Vol. 26, Na 7 (1991)

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543

water (3.3 mL) was added to stop the reaction. The prod- ucts were extracted with diethyl ether. The ether extract was washed five times with 2% aqueous sodium bicar- bonate, then washed with water, and dried over anhydrous sodium sulfate~ After removal of the solvent at ambient temperature, the 1- and 2-monoacylglycerol fractions were separated by preparative TLC on boric acid-impregnated silica gel plates (20 X 20 cn~ 0.5 mm thickness, boric acid 10% to Kiesel Gel 60G) developed in chloroform]methanol (98:2, v/v) under nitrogen.

Enantiomer separation of 1-monoacylglycerols by HPLC The 1-monoacylglycerol fractions (1-3 mg) were dissolved in dry toluene (500 ~L) and were reacted over- night at ambient temperature with 3,5-dlnitrophenyl iso- cyanate (10-20 nag) in the presence of dry pyridine (50 p~L). The chloroform solution of the products was spotted on a Kiesel Gel 60G plate and developed with n-hexane/ 1,2-dichloroethane/ethanol (40:15:6, v/v/v) under nitrogen. HPLC separations of the 1-monoacylglycerol 3,5-DNPU fractions were carried out using a Shimadzu LC-6A isocratic pump {Kyot~ Japan) with a SPD-1 ultraviolet spectrophotometric detecter and a C~R3A integrator from the same company. For optimum resolution, a chiral col- umn (25 or 50 cm • 4 nun i.&) packed with 5 ~an particles of N-(R)-l-(a-naphthyl)ethylaminocarbonyl-(S)-valine chemically-bonded to ),-aminopropyl sflAnlzed silica, Snmi- chiral OA-4100 (Sumitomo Chemical Ca) was used. The HPLC separation was done isocratically using a mixture of n-hexane/1,2-dichloroethane/ethanol (40:12:3, v/v/v) as mobile phase at a constant flow rate~ The 1-monoacyl- glycerol 3,5-DNPU sample was dissolved in the minimum volume of chloroform for injection. Detection was at 254 nm.

GC analysis of methyl esters. Total triacyl-sn-glycerols, 1- and 2-monoacylglycerols (1-2 rag), and the 3,5-DNPU derivatives of sn-1 and sn-3 monoacylglycerols (0.1-0.3 rag) were placed into 10 mL centrifuge tubes. Two mL of diethyl ether and 1 M sodium methoxide/methanol solu- tion {25 ~L) were added. The content was gently shaken for 5 rain before glacial acetic acid (6/~L) was added to stop the reaction. After removing the precipitate by cen- trifuging for 2 rain at 2000 rpm, and evaporating the sol- vent, the products were extracted with n-hexane and in- jected onto a WCOT column (50 m X 0.25 mm i.d., 0.2 pun film thickness) coated with Silar 5CP in a Shimadzu GC~6AM instrument with a FID detector and a Shimadzu C-R6A integrator. The column temperature was 190~ and the injector and detector temperatures were 230~ Hydrogen was the carrier gas.

RESULTS A N D D I S C U S S I O N

HPLC resolution of 1-monoacylglycerols. Enantiomer separations of 1-monoacylglycerol homologue mixtures of 3,5-DNPU derivatives by HPLC on a chiral stationary phase are accompanied by peak separations according to the number of carbon atoms and olefinic bonds of the com- ponents because of the polar nature of the chiral sta- tionary phase (8,10). In our previous paper (10), conditions for the resolution of enantiomers were reported that allow the separation of sn-1 and sn-3 groups of peaks. This was achieved mainly by using Sumichiral OA-4100 columns instead of the OA-2100, and by using less 1,2-dichloro- ethane in the eluent, i.e., a n-hexane/1,2-dichloroethane/

ethanol mixture (I0). In the present study, l-monoacyl- glycerol mixtures were obtained from triacyl-sn-glycerols of natural fats by partial hydrolysis with ethyl magnesium bromide The 1-monoacylglycerol mixtures were separated into sn-1 and sn-3 enantiomer groups by HPLC on the OA-4100 column. The 1-monoacylglycerols derived from vegetable oils containing palmitic, stearic, oleic, linoleic and linolenic acids were separated into sn-1 and sn-3 enan- tiomer groups by HPLC using a 25 cm column and n- hexane/1,2-dichloroethane/ethanol {40:12:3, v/v/v) as mobile phase at ambient temperatur~ Figure i shows the HPLC resolution of the 1-monoacylglycerols prepared from soy- bean oil triacyl-sn-glycerols. Complete resolution of race- mates into enantiomer groups was achieved for the 1- monoacylglycerol derivatives from soybean oil, maca- damia nut oil, and yew seed oil triacyl-sn-glycerols even when using micro-preparative HPLC. The time requLred for the separation was less than 30 rain. The sample amounts were about 2 and 200 pg per injection in analytical and micrc~preparative HPLC, respectively. The 1-monoacylglycerols from high erucic acid-rapeseed tri- acylglycerols required the 50 cm column for the clear separation of the enantiomers under similar conditions.

HPLC of fish oil 1-monoacylglycerols. In HPLC of 1-monoacylglycerols on an OA-4100 chiral column, the components eluted in the order of decreasing acyl chain- length, while a double bond in the acyl chain lengthened the retention time by the equivalent of about 2 carbon atoms (10). Thus, the retention time of an sn-1 18:3 monoacylglycerol derivative was similar to that of an sn-1 12:0 monoacylglyceroL These were the last components to emerge in HPLC of the 1-monoacylglycerol derivatives from plant oils reprsenting the sn-1 group, By the modified HPLC method (10), it was possible to separate these com- ponents from the first eluting components representing the sn-3 group, namely the sn-3 20:0 and 22:1 monoacyl- glycerol derivatives. However, when fish oils were used, complete separation of enantiomeric groups was more difficult. The first peak in the sn-3 group, Le., 22:1 or 24:1 monoacylglycerol derivatives, and the last peak in the sn-1 group, i.a, 16:4 or 22:6 monoacylglycerol deriva- tives, overlapped on the OA-4100 co | -m- at ambient

s__~n- I S_.D_n- 3 l J[ ]

18:1 16: i i 8:2 18:!

l / / 16:0 18:2 AA 18:0J \18:3 / v /

_ / .... ! i i i I i i

4 8 1 2 16 20 24 min

FIG. 1. Separation of 1-monoacylglycerols, formed by partial hydrolysis of soybean oil triacyl-sn-glycerols with ethyl magnesium bromide, as their 3,5-DNPU derivatives on a chiral col,mn, OA-4100 (25 cm X 4 mm i.d.). Mobile phase, n-bexane/12.-dichloroethanebtbenol (40:12:3, v/v/v). Flow rate, 1.0 mL/min; temperature, ambient; detec~ tion, 254 am UV.

LIRD& Vot. 26, NO. 7 (1991)

544

METHODS

temperatur~ We observed that lowering the col-ran temperature brought about a considerable improvement in enantiomer separatiom Figure 2 shows a chromatogram obtained at -9~ using 50 cm column. The separation factor for the enantiomer resolution increased from about 1.4 to about 1.8 by lowering the column temperature from 20~ to -9~ The time required for the separation of sn-1 and sn-3 monoacylglycerols from fish oils was about 200 mim

Differential losses in the conversions. Selective loss of monoacylglycerols in the preparation of 3,5-DNPU derivatives and methyl esters causes errors. To determine selective losses in the conversions, unsaturated 1-mono- acylglycerols (AI were converted to 3,5-DNPU derivatives {BI, and then to methyl esters (C) by the method described previously. In the present study, the following three samples were used {as AI in mixture with 1-stearoylglyc- erol: 1-oleoyl-, 1-linoleoyl- and 1-linolenoylglycerols {I); 1-arachidonoyl- and 1-eicosapentaenoylglycerols {II); and 1-docosahexaenoylglycerol (III). In each case, the results of HPLC of B and of GC of C were compared with the result of GC of the methyl esters obtained from A (Table 1). HPLC analysis of the 1-monoacylglycerol 3,5- DNPU was done as described in the previous paper (10). The results showed, in general, increasing losses as the unsaturation of the acyl groups increased. The apparent losses of methyl esters of unsaturated fatty acids could have been due to the short methylation t ime 3,5-DNPU derivatives prepared from sample I were therefore con- verted to fat ty acid methyl esters by transesterification in 1% sulfuric acid/methanol at 50~ overnight in a stop- pered centrifuge tub~ However, the fat ty acid composi- tion obtained by the latter method was close to that ob- tained by base-catalyzed transesterification as employed hem

Acyl compositions in sn-1, sn-2 and sn-3 positions. The composition (mole %) of the acyl group in each of the three positions was obtained by the following calculations us- ing the procedure outlined in Scheme 1. The mean com- position of each acyl group in the sn-1 and sn-3 positions [sn-l(3) position ] was calculated from the GC analysis of

40

s__qn - I s._~n - 3

i II I

.J

80 120 160 200

L rain

FIG. 2. Separation of 1-monoacylglycerols, formed by partial hydrolysis of herring oil triacyl-sn-glycerols with ethyl magnesium bromide, as their 3,5-DNPU derivatives on a chiral coh,m., OA-4100 (50 cm X 4 m m i~l.). Moldle phase, n-hexane/l~edichloroethane~thanol H0:12:3, v/v/v). Flow rate, 0.5 mL/min; temperature, --9~ detection, 254 um UV.

TABLE I

Conversion of Unsaturated 1-Monoacylglycerols to 3,5-DNPU Derivatives and Methyl Esters

Acyl Conversion to a

group 3,5-DNPU b Methyl ester c

18:0 1.000 1.000 18:1 1.014 0.998 18:2 0.972 0.913 18:3 1.041 0.900 20:4 0.905 0.815 20:5 0.865 0.765 22:6 0.885 0.667

a Change of ratios of unsaturated acyl groups to 18:0 acyl group in the conversion of 1-monoacylglycerols to 3,5-DNPU derivatives and methyl esters.

bAnalyzed by HPLC using the procedure described previously (10). c Analyzed by GC using the present procedure.

the original triacylglycerols and those of the 1- and 2-monoacylglycerols isolated by boric acid-impregnated silica gel TLC, i.e, for each component:

sn-l(3) position {mole %) = 3 x TG X [1-MG/(2 X 1-MG + 2-MG)]

where TG, 1-MG and 2-MG represent the proportions (mole %) of each acyl group in the original triacylglycerols, 1-monoacylglycerols and 2-monoacylglycerols, respec- tively. The composition of each acyl group in the sn-2 posi- tion was calculated in a similar manner as above, ie.:

sn-2 position {mole %) = 3 x TG X [2-MG/(2 X 1-MG + 2-MG)]

The amount of each acyl group in the sn-1 and sn-3 posi- tions was calculated from the concentration of each com- ponent in the sn-l(3) position and GC analyses of sn-1 and sn-3 monoacylglycerols resolved by HPLC on the chiral column, i.~:

sn-1 position {mole %) = 2 X sn-l(3) position X [sn-ll(sn-1 + sn-3)]

sn-3 position {mole %) = 2 X sn-l(3) position X [sn-31(sn-1 + sn-3)]

where sn-1 and sn-3 represent the proportions (mole %) of each acyl group in sn-1 and sn-3 monoacylglycerols analyzed by GC as methyl esters, respectively. These calculations avoid cumulative errors in the result for a par ~ ticular positio~ and give reproducible and reliable results. In contrast, direct analysis of methyl esters from sn-1, sn-2 and sn-3 monoacylglycerols can involve considerable ex- perimental errors. The composition of the acyl group in each of the three positions could also be obtained based on the following relationships:

sn-1 position = 3 X TG - 2-MG -- sn-3

sn-2 position = 3 X TG - sn-1 - sn-3

sn-3 position = 3 X TG - sn-1 - 2-MG

However, this approach is not recommended, since the result for each position is subject to the cumulative ex- perimental error of the other analyses.

UPIDS, Vol. 26, No. 7 (1991)

METHODS

545

TABLE 2

Reproducibility of Positional Distributions of Acyl Groups in Triacyl-sn-Glycerols from Soybean Oil (mole %)

Acyl group mole percent Acyl and standard deviation (n--4)

No. group Triacylglycerol sn-l(3) a sn-1 sn-2 sn-3

1 14:0 0.1 0.1 b • 0.0 c 0.1 • 0.0 0.2 • 0.0 0.1 2 16:0 11.6 16.0 • 0.3 16.6 • 0.2 3.0 • 0.6 15.5 3 16:1n-7 0.1 0.1 • 0.0 0.1 • 0.1 0.1 • 0.0 0.1 4 17:0 0.1 0.1 • 0.0 0.1 • 0.0 0.1 • 0.0 0.1 5 18:0 3.4 4.8 • 0.1 4.9 • 0.3 0.6 • 0.2 4.7 6 18:1n-9 22.7 22.6 • 0.1 22.4 • 0.1 23.0 • 0.2 22.7 7 18:1n-7 1.4 1.9 ___ 0.0 1.9 • 0.1 0.4 • 0.1 1.9 8 18:2n-6 53.9 47.2 • 0.3 46.8 • 0.3 67.0 • 0.7 47.7 9 18:3n-6 6.4 6.8 • 0.1 6.7 • 0.2 5.7 -- 0.1 6.9

10 20:0 0.3 0.5 • 0.0 0.5 • 0.1 ND d 0.4

--. 0.0 • 0.6 • 0.1 • 0.0 • 0.1 • 0.2 • 0.1 • 0.5 • 0.2 • 0.0

a Primary position in triacyl-sn-glycerol. bMean value of quadruplicate analyses. c Standard deviation. dND, not detected.

TABLE 3

Comparison of Stereospecifie Distributions of Acyl Groups in Soybean Oil Trlacyl-sn-Glycerols Obtained by HPLC and Enzymatic Methods

Composition (mole %)

HPLC method a Enzyme methodb Acyl No. group sn-1 sn-2 sn-3 sn-1 sn-2 sn-3

1 14:0 0.1 0.1 0.1 0.3 0.3 - 0 . 2 2 16:0 16.7 2.2 16.1 16.9 1.4 16.6 3 16:1no7 0.1 0.1 0.1 0.1 0.1 0.0 4 17:0 0.1 0.0 0.1 0.1 0.0 0.0 5 18:0 5,4 0.3 4.6 5.2 0.3 4.7 6 18:1n-9 22,2 23.0 22.9 20.3 22.6 25.1 7 18:1n-7 2.1 0.4 1.7 2.0 0.4 1.8 8 18:2n-6 46.4 68.1 47.0 47.4 69.2 45.2 9 18:3n-6 6.4 5.7 7.0 7.5 5.7 5.9

10 20:0 0.5 ND c 0.4 0.2 ND 0.7

aMethod presented here. bModified Brockerhoff method (6). c ND, not detected.

Reproducibil i ty o f the stereospecific analysis. The reproducibility of the analytical method was checked for soybean oil triacyl-sn-glycerols. Table 2 shows the results. The stereospecific analyses carried out by four different persons showed good agreement. Another soybean oil triacyl-sn-glycerol sample was analyzed by the present method and by the Brockerhoff method as modified by Christie {6). The results are compared in Table 3. The posi- tional distributions obtained differed little from each other.

Acy l migration in the course of analysis. Monoacylglyc- erols generally isomerize more rapidly than do diacyl- glycerols due to acyl migration. Isomerization affects the accuracy of a method. The accuracy of our method was determined by the stereospecific analysis of racemic syn- thetic triacyl-sn-glycerols {Table 4}. The results showed

that the sn-1 and sn-3 positions can be cont~mlnated with 2.5-3.0% of acyl groups which have migrated from the sn-2 position. This would i nd i ca t e tha t the error in the relative proportions at the sn-1 and sn-3 positions could be about 1%, when the acyl group in sn-2 position is 30% of the total. On the other hand, the sn-2 position contain- ed 4-6% of acyl groups tha t migrated from the sn-1 and sn-3 positions {Table 4). However, the total acyl composi- tions suggest tha t the 1,3-dioleoyl-2-stearoylglycerol and 1,3-dipMmltoyl-2-1inolenoylglycerol samples contained 1.5% 18:1 acyl and 5.1% 16:0 acyl in the sn-2 position, respectively. Thus, the sn-2 position can be contaminated with up to 3.0% of acyl groups which have migrated from the pr imary positions. These results demonstrate the relatively low rate of isomerization of monoacylglycerols, as compared to what has been reported for diacylglycerols

UPIDS, Vol. 26, No. 7 (1991)

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METHODS

TABLE 4

Stereospedfic Analysis of Synthetic Triacyl-sn-Glyeerols to Examine Isomerization in the Procedure

Structure Acyl Composition (mole %) of sample group Total sn-l(3} a sn-1 sn-2 sn-3

18:1-18:0-18:1 18:0 32.8 2.8 2.6 95.8 3.0 18:1n-9 67.2 97.2 97.4 4.2 97.0

16:0-18:3-16:0 16:0 68.4 97.5 97.6 6.1 97.5 18:3n-3 31.6 2.5 2.4 93.9 2.5

aprimary position in triacyl-sn-glycorol.

TABLE 5

Stereospecific Analysis of Macadamia Nut Triacyl-sn-Glycerois

Acyl Composition (mole %)

No. group Total sn-1 sn-2 sn-3

1 12:0 0.4 ND a 0.4 0.8 2 14:0 1.4 1.3 1.0 1.9 3 16:0 9.0 12.5 3.1 10.8 4 16:1n-7 20.2 23.4 17.2 18.2 5 16:1n-5 0.3 4.3 3.0 2.0 6 18:0 3.1 3.2 0.6 5.3 7 18:1n-9 56.3 47.9 71.0 45.1 8 18:1n-7 3.4 4.5 1.3 4.0 9 18:2n-6 1.5 1.7 2.0 0.5

10 18:3n~ 0.1 ND 0.4 ND 11 20:0 2.2 0.3 0.0 6.1 12 20:1n-9 2.1 1.0 " ND 5.2

aND, not detected.

{5,12}. For example, 1,3-diacylglycerols prepared by reac- tion with ethyl magnesium bromide contained 7-10% {or more} of acyl groups that had migrated from the sn-2 posi- tion {5,12}. The sn-2 position of 1,2-diacylglycerols was contemi~ated with 4% of acyl groups that had migrated from the sn-1 and sn-3 positions (5,12}. The stereospecific analysis of the natural triacyl-sn-

glycerols also supports the conclusion that acyl migration is minimal in the present method. Triacyl-sn-glycerols from macadaqmia nut oil, yew seed oil, high erucic acid r a p e s e e d oil a n d h e r r i n g oil were s u b j e c t e d to stereospe~ cific a n a l y s i s {Tables 5-8}. F a t t y ac id c o m p o s i t i o n s of m a c a d a m i a n u t and yew seed oils have been r e p o r t e d p r ~ v i o u s l y {13,14}. M a c a d a m i a n u t oil t r i acy l - sn -g lyce ro l s showed 20:1n-9 acy l g roups a t an a b u n d a n c e of 5.2% in t he sn-3 pos i t ion and of 1.0% in t he sn-1 posi t ion, b u t these g r o u p s were n o t d e t e c t e d in t h e sn-2 p o s i t i o n {Table 5). Yew seed oil t r i acy l - sn-g lycero ls showed all-cis-5,9,12-18:3 a n d 5,11,14-20:3 acyl g r o u p s a t c o n c e n t r a t i o n s of 8.1 a n d 4.7%, respect ive ly , in t h e sn-3 pos i t ion , b u t t h e s e acyl g roups were e s s e n t i a l l y a b s e n t in t h e o t h e r p o s i t i o n s {Table 6}. The r apeseed oil t r i acy l - sn-g lycero ls showed the 22:1n-9 acy l g r o u p a t c o n c e n t r a t i o n s of 61.3% a n d 50.2% in t he sn-3 a n d the sn-1 posi t ions , respect ively, b u t 22:1n-9 was on ly 0.8% a b u n d i m t in sn-2 p o s i t i o n {Table 7). H e ~ r i n g oil t r i acy l - sn-g lycero ls showed t h e 22:6n-3 acyl g roup a t c o n c e n t r a t i o n s of 14.9% in t h e sn-2 p o s i t i o n a n d of

TABLE 6

Stereospecific Analysis of Yew Seed Triacyl-sn-Glycerols

Composition (mole %) Acyl

No. group Total sn-1 sn-2 sn-3

1 16:0 2.7 5.7 1.7 O.~ 2 16:1n-9 0.1 0.0 0.1 0.0 3 18:0 1.4 3.7 0.4 0.2 4 18:1n-9 36.7 50.0 52.9 9.0 5 18:1n-7 0.6 0.2 0.3 1.2 6 18:2(5,9) 18.5 1.7 1.7 50.0 7 18:2n-6 31.6 30.8 41.5 23.3 8 18:3(5,9,12) a 2.9 0.2 ND b 8.1 9 18:3n-3 1.3 1.5 1.3 1.2

10 18:4(5,9,12,15) 0.2 ND 1.2 0.2 11 20:1n-9 1.5 4.2 ND 0.4 12 20:2n-6 1.0 2.0 ND 0.9 13 20:3(5,11,14) 1.6 ND ND 4.7

a All-cis-5,9,12-octadecatrienoyl group. Other 5-olefinic acyl groups also have the cis configuration.

bND, not detected.

TABLE 7

Stereospecific Analysis of Rapeseed Triacyl-sn-Glycerols

Acyl Composition (mole %)

No. group Total sn-1 sn-2 sn-3

1 14:0 0.1 0.1 0.2 0.1 2 16:0 3.9 5.6 1.6 4.5 3 16:1n-9 0.1 ND a 0.1 0.6 4 16:1no7 0.3 0.3 0.3 0.2 5 17:1no8 0.1 ND 0.3 ND 6 18:0 1.3 1.9 0.4 1.7 7 18:1n-9 17.2 13.7 32.4 5.1 8 18:1n~ 1.4 2.8 1.0 0.5 9 18:2n-6 16.1 1.2 39.3 7.4

10 18:3n-3 8.7 3.3 21.3 1.3 11 20:0 0.8 0.6 0.3 1.5 12 20:1n-9 8.9 15.5 1.5 10.0 13 20:1n-7 1.5 1.9 0.4 2.0 14 20:2n-6 0.5 1.2 0.1 0.2 15 22:0 0.7 ND ND 2.1 16 22:1n-9 37.4 50.2 0.8 61.3 17 22:1n-7 0.6 0.9 ND 1.0 18 22:2n-6 0.5 0.9 ND 0.7

aND, not detected.

2.19% in t h e sn-1 pos i t ion , b u t 22:6n-3 was n o t d e t e c t e d in t he sn-3 pos i t i on (Table 8). Such spec i f ic i ty in m e a s u r e d pos i t i ona l d i s t r i b u t i o n ind i ca t e s t h e low degree of isomer- i za t i on of m o n o a c y l g l y c e r o l s in t h e p roc e du re s of s tereo- speci f ic a n a l y s i s e m p l o y e d h e m

Selec t iv i ty o facy l distributions. A s a genera l rule, seed oils con ta in ing c o m m o n f a t t y ac ids show a select ive p lace ~ m e n t of u n s a t u r a t e d f a t t y ac ids a t t he sn-2 posi t ion , while long-cha in or a n o m a l o u s f a t t y ac ids a re se l ec t ive ly en- r i ched in t h e sn-3 p o s i t i o n {7}. Table 5 shows t h a t t h e 18:1n-9 g r o u p was en r i ched (c~ 71%) a t t h e sn-2 pos i t ion , b u t t h a t 18:1n-7 was more a b u n d a n t a t t h e sn-1 and sn-3 posi t ions. Th is shows t h a t t he pos i t ion of t he olefmic b o n d

LIPIDS, Vol. 26, Na 7 0991)

METHODS

547

TABLE 8

Stereospecifie Analysis of Herring Triacyl-sn-Glycerols

Acyl Composition (mole %)

No. group Total sn-1 sn-2 sn-3

1 14:0 9.7 11.9 6.7 9.7 2 4,8,12-TMTD a 0.3 0.1 0.4 0.5 3 is~15:0 0.1 0.1 0.2 0.1 4 15:0 0.6 0.4 0.9 0.5 5 16:0 15.7 15.2 17.9 12.5 6 16:ln-ll 0.3 0.5 0.3 ND b 7 16:1n-9 0.2 0.1 0.5 0.1 8 16:1n-7 5.0 6.4 3.8 4.3 9 iso-17:0 0.5 0.6 0.4 0.4

10 17:0 0.6 1.4 0.3 ND 11 16:2n-4 0.4 0.5 0.6 0.1 12 16:3n-4 0.3 0.1 0.6 0.2 13 16:4n-1 0.4 0.9 0.4 ND 14 18:0 0.9 0.9 0.9 0.8 15 18:1n-9 9.9 11.3 8.7 9.0 16 18:1n-7 1.5 2.4 0.8 1.1 17 18:1n-5 0.5 0.6 0.5 0.3 18 18:2n-6 1.2 1.2 1.4 1.0 19 19:0 0.2 0.5 0.1 ND 20 18:3n-3 1.4 1.2 1.6 1.2 21 18:4n-3 3.8 4.6 3.9 2.4 22 20:0 0.1 0.1 0.1 0.2 23 20:ln-ll 1.2 1.4 0.6 1.6 24 20:1n-9 11.8 16.3 4.2 14.4 25 20:2n-6 0.2 0.1 0.1 0.4 26 20:4n-6 0.2 0.0 0.2 0.2 27 20:4n-3 0.5 1.0 0.5 ND 28 20:5n-3 5.9 3.0 10.5 3.3 29 22:ln-ll,n-13 16.0 12.3 5.2 31.6 30 22:1n-9 0.8 0.2 1.5 0.5 31 21:5n-3 0.3 0.3 0.5 ND 32 22:5n-3 0.5 0.2 1.2 ND 33 22:6n-3 6.1 2.2 14.9 ND 34 24:1n-9 0.7 0.5 0.3 1.4

a 4,8,12-TMTD, 4,8,12-trimethyltetradecanoyl group. bND, not detected.

is an important factor in governing acyl distributions as is the number of carbon atoms and the number of double bonds. Table 6 shows tha t anomalous 5-olefinic acyl groups were selectively concentrated in the sn-3 position as predicted (7). Their accumulation at the sn-3 position, in turn, resulted in a lower relative proportion of 18:1n-9 at this positiom Table 7 shows the positional distributions of 18 different acyl groups in rapeseed oil triacyl-sn- glycerols. The characteristic distribution patterns of seven components of acyl groups in triacyl-sn-glycerols from seeds of nine Cruciferae, as determined by Brockerhoffs method, were reported previously (15). The data presented in Table 7 are in generally good agreement with these in as much as 18:1n-9 was enriched at the sn-2 position, whereas 18:1n-7 was more abundant in the sn-1 position. Table 8 shows the distributions of 34 acyl groups of

herring oil triacyl-sn-glycerols. The distributions of 11 acyl groups in herring oil had previously been reported by Brockerhoff et aL {16). The 11 most abundant components {16) amount to about 80% of acyl groups listed in Table 8. The method presented here is especially useful for the analysis of the complex acyl group compositions of the triacyl-sn-glycerols of marine lipide.

Comparison wi th the Brockerhof f method. In the Brockerhoff method the composition at the sn-3 position is not determined directly. So errors can occur particularly for minor components at the sn-3 position, and sometimes small negative values may be obtaineck Another drawback can be the preferential hyrdolysis of certain acyl groups by phospholipase As, which affects the data for the sn-2 position. The most common source of phospholipase A2 for structural studies are snake venoms. King cobra, Ophiophagus hannah, venom is preferred because it had been shown not to preferentially hydrolyze 16:0, 18:0, 18:1, 18:2 and 20:0 from lecithins (17). I t has therefore been used for the stereochemical analysis of triacylglycerols. �9

However, triacyl-sn-glycerols from natural sources con- tain a great variety of acyl groups, and there is at pres- ent insufficient evidence for the nonpreferential hydrolysis of all types of acyl groups.

The method presented here permits the stereospecific analysis of triacyl-sn-glycerols. The procedures used are simple and rapid, and permit measurement of distribu- tions even of minor components when high resolution capillary GC is employed.

REFERENCES 1. Brockerhoff, H. (1965} J. Lipid Res. 6, 10-15. 2. Lands, W.E.M., Pieringer, R.A., Slakey, RM., and Zschocke, A.

{1966) Lipide 1, 444-448. 3. Brockerhoff, H. (1967} J. Lipid Res. 8, 167-169. 4. Myher, J.J., and Kuksis, A. (1979} Can. J. Bioehem. Cell. BioL

57, 117-124. 5. Christie, W:W., and Moore, J.H. (1969) Biochim. Biophys. Acta

176, 445-452. 6. Christie, W:W. (1982) Lipid Analysis, 2nd edn., pp. 158-161,

Pergamon Press, Oxford. 7. Breckenridge, W.C { 1978) in Handbook of Lipid Research (Kuksis,

A., ed.) VoL 1, pp. 197-232, Plenum Press, New York. 8. Takagi, T., and Ando, Y. (1990) J. Japan. Oil Chem. Soa 39,

622-628. 9. Laakso, P., and Christie, W:W. (1990) Lipids 25, 349-353.

10. Tabagi, T., and Ando, Y. (1990) Lipids 25, 398-400. 11. Bligh, E.G., and Dyer, W.J. (1959) Cam J. Biochem. PhysioL 37,

911-917. 12. Brockerhoff, H. (1971) Lipids 6, 942-956. 13. Vickery, J.R. (1971) Phytoehemistry 10, 123-130. 14. Takagi, T., and Itabashi, Y. (1982) Lipids 17, 716-723. 15. Ohlson, R., Podlaha, O., and Toregard, B. (1975) Lipide 10,

732-735. 16. Brockerhoff, R.J., Hoyle, R.J., Hwang, PC., and Litchfield, C.

(1968) Lipide 3, 24-29. 17. Nutter, L.J., and Privett, O.S. (1966) Lipids 1, 258-262.

[Received September 5, 1990, and in revised form January 31, 1991; Revision accepted April 20, 1991]

LIPIDS, Vol. 26, Na 7 0991)