Structural Analysis of Hydroperoxides Formed

by Oxidation of Phosphatidylcholine
with Singlet Oxygen
J. TERAO, Y. HIROTA, M. KAWAKATSU and S. MATSUSHITA, Research Institute
for Food Science, Kyoto University, Uji, Kyoto, 611, Japan

ABSTRACT
Soybean phosphatidylcholine (PC) and dilinoleoyl PC (di-18:2 PC) were oxidized with singlet
molecular oxygen using methylene blue as the photosensitizer. The oxidation products, PC mono-
hydroperoxides (PC-MHP) and PC dihydroperoxides (PC-DHP), were isolated by reverse phase liquid
chromatography, and their structures were analyzed by nuclear magnetic resonance (NMR) and gas
chromatography-mass spectrometry (GC-MS). Signals for the hydroperoxy proton appeared downfield
in NMR spectra of PC-MHP and PC-DHP. Soybean PC-MHP and di-18:2 PC-MHP were converted to
trimethylsilyl (TMS) derivatives of hydrogenated diglycerides when treated with phospholipase C and
hydrogenated. The tert-butyldimethylsilyl (TBDMS) derivatives of hydrogenated diglycerides were
also prepared from di-18:2 PC-MHP. Fragmentation of the TMS and TBDMS derivatives was obtained
in electron impact mass spectra. The isomeric composition of hydroperoxylinoleate component in
di-18:2 PC-MHP was determined by methanolysis of the hydrogenated diglyceride and mass chromato-
graphic analysis of the resulting isomeric hydroxy octadecanoates.

iNTRODUCTION hydrogenated. GC-MS analysis of the saturated

Peioxidation of membrane phospholipids
has been suggested as causing physiological
damage in living organisms (1,2). Polyunsatu-
rated fatty acids (PUFA) constituting phospho-
lipids are susceptible to oxidation and produce
hydroperoxides as the primary oxidation
products (3). Hydroperoxides formed by free
radical oxidation of PUFA have been analyzed
using high performance liquid chromatography
(HPLC) (4-6) and gas chromatography-mass
spectrometry (GC-MS) (7-10). Recently, HPLC
was applied to the separation of oxidized and
unoxidized phosphatidylcholine (PC) molecular
species (11). Porter et al. (12) succeeded in
isolating PC hydroperoxides produced by free
radical oxidation of unsaturated PC and used
HPLC to characterize the oxygenated fatty acid
constituents.
On the other hand, the role of singlet
molecular oxygen (102) in lipid peroxidation
has been widely discussed (13-17). This active
oxygen molecule reacts with unsaturated fatty
acid producing isomeric hydroperoxides (18-
21). The isomeric compositions of monohydro-
(oeroxides formed by oxidation of PUFA with
2 have already been determined by GC-MS
analysis (22-24).
This paper reports on the oxidation of
soybean PC and dilinoleoyl PC (di-18:2 PC) by
102 using a methylene blue-sensitized photo-
oxidation system (20), and the oxidation
products, PC monohydroperoxides (PC-MHP)
and PC dihydroperoxides (PC-DHP). PC-MHP
were converted to saturated diglyceride deriva-
tives when treated with phospholipase C and
diglycerides was performed after trimethylsilyl-
ation or tert-butyldimethylsilylation. Isomeric
composition of the h y d r o p e r o x y fatty acid
component of di-18:2 PC-MHP was determined
by mass chromatographic analysis. The reaction
mechanism of unsaturated PC with 102 is
discussed.
EXPERIMENTAL PROCEDURE
Materials
Soybean PC purchased from Nakarai Chem.
Co. Ltd., Kyoto, Japan, was washed before use
with acetone and purified by silica gel column
chromatography (25). Linoleic anhydride was
synthesized from the reaction of linoleic acid
(99%, Nakarai Chem. Co. Ltd.) with dicyclo-
hexycarbodiimide in dry carbon tetrachloride
(26). Glycerophosphorylcholine-cadmium com-
plex (GPC-CdCI2) was prepared from purified
soybean PC according to the method of Chadha
(27). Dilinoleoyl PC (di-18:2 PC) was synthe-
sized by 1,2-diacylation of the GPC-CdC12
complex with linoleic anhydride according to
the method of Patel et al. (28). Phospholipase
C (EC 3.1.4.3) from Clostridiurn perfringens
was obtained from P-L Biochemicals, Milwau-
kee, WI. Trimethylsilyl (TMS)/pyridine reagent
and tert-butyldimethylsilyl (TBDMS)/imidazole
reagent were purchased from T o k y o Kasei
Kogyo, Tokyo, Japan, and Applied Science
Labs, State College, PA, respectively.
Photooxiflation Procedure
PC (100 mg) was dissolved in 5 ml of meth-

427
428 J. TERAO, Y. HIROTA, M. KAWAKATSU AND S~ MATSUSHITA
anol containing 0.1 mM of methylene blue. A
reaction vessel holding the solution was placed
in a water bath (25 C) and shaken continuously
with illumination of a 30-W tungsten projection
lamp for 12 hr (intensity at the sample; 10 mW/
c m 2 ).
Isolation of PC Hydroperoxides
After photooxidation was completed, the
reaction mixture was concentrated and applied
to a reverse phase glass column (240 x 10 mm)
prepacked with Lichloprep RP-8 (Merck,
Darmstadt, silica gel powder binding octane,
40-63 /am size) and eluted with chloroform/
methanol/water (1:10:0.5, v/v). Solvent flow
was maintained at 1.8 ml/min and 1.0-ml
fractions collected. Phosphorus content (29)
and absorbance at 235 nm were determined for
each fraction. The fractions with oxidation
products were collected and concentrated in
vacuo. Nuclear magnetic resonance (NMR)
spectra were obtained in carbon tetrachloride
with a Hitachi-Perkin Elmer Model 90 (90
MHz).
Derivatization
PC-MHP (30 mg) was dissolved in 3 ml of
ethyl ether/ethanol (98:2). To this solution was
added 0.5 ml of tris buffer (10 mM, pH 7.4)
containing CaCI2 (20 mM) and 5 mg of phos-
pholipase C (1-2 unit/mg protein). The solution
was shaken during the reaction for 15 min at
30 C. After the reaction was completed, the
ethyl ether layer was evaporated in vacuo. The
residue was dissolved in ethanol and hydro-
genated with palladium on carbon in a stream
of hydrogen. Trimethylsilylation and tert-
butyldimethylsilylation of the hydrogenated
derivatives were performed by heating with
TMS reagent at 60 C for 5 min, and with
TBDMS reagent at" 160 C for 10 min, respec-
tively.
120 B 14.4%; stearic acid (18:0), 3.2%; oleic acid
2D~
,.~80
20 4o eo
froct k.n number
FIG. 1. Reverse phase liquid chromatography of
oxidized soybean PC. - - : phosphorus content; ..... :
absorbance at 235 nm after dilution with ethanol.
LIPIDS, VOL. 16, NO. 6 (1981)
GC-MS
A system of GC-MS PAC 300, consisting of
a Shimdzu LKB-9000 spectrometer and OKI-
TAC 4300S minicomputer, was used. The
column was a glass spiral tube (0.35 m • 3
mm), packed with 2% OV-1 on Neopack 1A,
60/80 mesh. Helium gas was used at 30 ml/min.
Temperature of the oven was programmed from
260 to 290 C (6 C/min). Operation conditions
for mass spectrometer were: ion source temper-
ature, 310 C; separator temperature, 300 C;
ionizing electron energy, 22 eV, trap current,
60/2A; and accelerator voltage, 3.5 kV.
Analysis of Fatty Acid Composition
and Molecular Species of Soybean PC
Fatty acid composition of soybean PC was
determined by gas liquid chromatography
(GLC) after methanolysis using sodium meth-
oxide in methanol solution. For GLC, a Shimd,
zu GC-5A was used with a glass column (2.5 m
x 3 mm) packed with 15% DEGS on Neopack
AS, 60/80 mesh. The flow rate of nitrogen gas
was 60 ml/min, and column oven temperature
was 187 C. Molecular species of soybean PC
were analyzed according to the method of
Nishihara and Kito (30).
Determination of Isomeric Composition
of Hydroperoxylino|eate Component
in Di-18:2 PC-MHP
Hydrogenated diglyceride prepared from
di-18:2 PC was subjected to methanolysis.
Isomeric composition of methyl hydroxyocta-
decanoate derived from hydroperoxylinoleate
component of PC-MHP was determined by mass
chromatographic analysis according to the
method of Frankel et al. (22). The conditions
for mass chromatography were the same as
described previously (23).
R ESU LTS
Fatty acid composition of soybean PC was
determined as follows: palmitic acid (16:0),
(18:1), 11.3%; linoleic acid (18:2), 64.8%; and
linolenic acid (18:3), 6.3%. Principal molecular
species of soybean PC were 16:0-18:2 (25.2%),
18:1-18:2 (13.2%), and di-18:2 (31.3%). The
percentages of other molecular species were
each less than 10%.
Figure 1 shows the result of reverse phase
liquid chromatography of photooxidized soy-
bean PC. Two peaks, A and B, appeared as the
oxidation products and were separated from
nonoxidized PC. Ultraviolet spectra of the
fractions, A and B, gave their XEtOxH at 235 nm
due to conjugated diene. In the NMR spectra
 PC OF HYDROPEROXIDES FORMED BY IO2 429

ba

B h

i
I 1 I I I I J I I J
12 10 8 6 4
ppm

FIG. 2. NMR spectra of fractions A and B. Signals were assigned to each proton as
follows: a, -CH3 ; b, -CH2 ; c, --~CH-CH2-; d, CH2CO; e, ~---CH-CH2-CH=; f, N(CH3)3 ; g,
CH2OCO, CH2N(CHa)3, CH2OPO, and CHOOH; h, CHOCO-, CH~-CH-C_H2; i, -CH=
CH-CH~---CH-;J, -HCOOH.

of the two fractions (Fig. 2), broad signals 2

downfield in the region of 10-13 ppm were
assigned to hydroperoxy protons. After addi-
tion of D20 to the NMR tube, the signals for
hydroperoxy protons diminished to the base
line. The number of protons in the region of
hydroperoxy protons were 1.6 for fraction A 1 soybean
and 0.5 for fraction B. GLC analysis of the
fatty acid components of the two fractions
after hydrogenation and methanolysis showed
that fraction A consisted of only monohy-
droxyoctadecanoate and fraction B consisted
of three components, i.e., monohydroxyocta-
decanoate, octadecanoate and hexadecanoate.
From these data, fraction A was identified as
\
PC-DHP consisting of two hydroperoxy fatty
acid components a n d fraction B, PC-MHP 3
consisting of one hydroperoxy fatty acid and
one unoxidized fatty acid component. Two di18:2
oxidation products were also obtained from
di-18:2 PC using the same procedure as for
soybean PC, and were identified as PC-DHP
consisting of two hydroperoxylinoleate com-
ponents, and PC-MHP consisting of one hydro-
peroxylinoleate and one unoxidized linoleate
component.
Figure 3 shows the gas chromatogram of the
TMS derivatives of hydrogenated diglycerides
derived from soybean PC-MHP and di-18-2
PC-MHP. Mass spectra of the three peaks (Fig. 6 s lb 1-g
4) show fragment ions characteristic for TMS rain
derivatives of diglycerides at m/z 129 and 145
(31,32), although the molecular ions, [M], FIG. 3. Gas chromatograms of the TMS derivatives
were not detected. In the spectrum of peak 1, of hydrogenated diglycerides obtained from PC-MHP.

LIPIDS, VOL. 16, NO. 6 (1981)

430 J. TERAO, Y. HIROTA, M. KAWAKATSU AND S. MATSUSHITA

! 129 [RICO+74] +
73 ] [RICO] + 30 ] [M-R' COO] +
[M-90] + [M-15]+

, . l,,, Ll
355 429 u'
. t,t L , , , t
I ,
666
,
I
741
k
I
0 i00 200 300 400 500 600 70O
.>'[~ 1129
"~ |-" / t"-R'coo] +
[, / / ~o~ R,oo~+ 41~,,' [.-15] I
"- ] 73 | + [ I x3
e-! t / 145 , 229 [R~co] 131~ 35~ I I [M-90x2]+604 [M-90]+694 ~
"~ | / I , ~i~F}51 2~r / I ~,1 :'" ,I |429 /--
'7- 400 500 600 700
129
[R2C0+74 ]+ 413
145173 301 ~ 343 x3
215229 969

0
I ],
I
.,tl~

i00
I I

200
, ,
I -, a

300
[_~3~5
I
a
I

400
I
I

50'9
I
604
it I
694
'I I t
600 700
mlz

FIG. 4. Mass spectra of peaks 1, 2 and 3. R l = CIsH3,-, R~ = C17H3s-, R~ = CmH34OSi(CHa)3-.

characteristic ions for the TMS derivative of 229 [(CHa)3SiOCH(CH2)sCH3 ], 215 [(CHa)3-
hexadecanoyl-hydroxyoctadecanoyl diglyceride SiOCH(CH2)TCH 3 ], 187 [ (CH 3 )3SiOCH(CH 2 )s"
were present at m/z 741 [M-15, loss of CH3], CH3], and 173 [(CH3)aSiOCH(CH2)4CH3].
666 [M-90, loss of trimethylsilanol], 576 These fragment ions seem to be yielded by
[666-90], 429 [C17HaaOSi(CH3)3CO + 74], a-cleavage of the trimethylsilyloxy group
385 [M-371, loss of C,TH~OSi(CH3)aCOO], producing a hydrocarbon fragment (20). The
355 [C17H34OSi(CH3)3CO], 313 [ClsHalCO other series of fragment ions at m/z 301 [C2Hs-
+ 74], and 239 [ClsH3,CO]. The spectra of OCOOC(CH2)TCHOSi(CH3)3], 315 [C2HsOC-
peaks 2 and 3 show characteristic ions for the OOC(CH2)sCHOSi(CH3)3], 343 [C2HsOCO -
TMS derivative of octadecanoyl-hydroxyocta- OC(CH2h0CHOSi(CHa)a] , and 357 [C2Hs-
decanoyl diglyceride at m/z 769 [M-15], OCOOC(CH2)nCHOSi(CH 3)3] is probably
694 [M-90], 604 [694-90], 429, 413 [M-371], derived from the a-cleavage, producing an ester
355, 341 [C17H35CO + 7 4 ] , and 267 [C17Has- fragment. Fragmentation of these four ions can
CO]. These fragmentation patterns are anal- be explained by elimination of the acyl and
ogous to those of TMS derivatives of digly- trimethylsilyloxy groups or acyloxy and
cerides (31,32). Thus, peak 1 was identified as trimethylsilyl groups from the a-cleavage ions.
the TMS derivatives of hexadecanoyl-hydroxy- The carbon numbers indicating the position of
octadecanoyl diglyceride and peaks 2 and 3, the the h y d r o x y group attached to the fatty acid
TMS derivatives of octadecanoyl-hydroxyocta- component are: m/z 229, 301 (9-), 215, 315
decanoyl diglyceride. Peaks 1 and 2 are presum- (10-), 187, 343 (12-) and 173, 357 (13-).
ably derived from different PC molecular Figure 5 shows the mass spectrum o f the
species, i.e., peak 1 from 16:0-18:2 and peak 2 TBDMS derivatives of hydrogenated diglyceride
from 18:1-18:2 and di-18:2. Fragment ions obtained from 18:2 PC-MHP. Fragment ions of
formed by elimination of the acyloxy group high intensity appeared at m/Lz 811 [M-57, loss
from molecular ion, [M-R1COO] or [M-R2COO] of C(CH3)3], 455 [M-413, loss of CtTH34OSi-
m/z 501, did not appear in the three spectra, (CH3)2C(CH3)3], and 171 [CH2CH-CHOSi-
although fragment ions formed by elimination (CH3)2C(CH3)3]. This fragmentation pattern
of the trimethylsilyloxyacyloxy group from is very similar to that of TBDMS derivatives of
molecular ion, [M-R2COO], were of signifi- diglycerides (33). The ions formed by elimi-
cantly high intensity (Fig. 4). nation of the acyloxy group, [M-RCOO],
In the mass spectra of the TMS derivatives m/z 575, were not detected in the spectrum as
of diglyceride from soybean PC-MHP and di- in the case of the TMS derivatives. It is prob-
18:2 PC-MHP (Fig. 4), fragment ions indicating able that elimination of the acyloxy group
the position of the h y d r o x y group attached to containing a silylated h y d r o x y group occurs
the fatty acid component were present at m/z predominantly during fragmentation of the

LIPIDS, VOL. 16, NO. 6 (1981)

 PC OF HYDROPEROXIDES FORMED BY t O 2 431

c-
a I I I I
.S 500 600 700 800
+ +
171 [M-R'COO]
[R'CO+74]
.g
[RCO] + 339
l [R'CO]
267 397 4
, ... LI L
[ I

10 200 500 qO0 500

m/z

FIG. 5. Mass spectrum of the TBDMS derivatives of hydrogenated diglyceride obtained

from di-18:2 PC-MHP. R = CtTH35-, R~= C17H~OSi(CHa)2C(CH3)3-.

TMS and TBDMS derivatives of diglycerides
from PC-MHP.
Mass chromatography of the hydroperoxy-
linoleate component of di-18:2 PC-MHP was
done after enzymatic hydrolysis, hydrogena-
tion, methanolysis and trimethylsilylation.
F o u r positional isomers, the 9-, 10-, 12-, and
13-isomers, were present in the resulting TMS
derivatives of methyl h y d r o x y octadecanoate
(Fig. 6). Thus, it is apparent that the four
positional isomers are present in the hydro-
peroxylinoleate component of di-18:2 PC-MHP.
The quantitative ratio of the isomeric hydro-
peroxylinoleate component was determined
from the peak areas in Figure 6 as follows:
9-:10-:12-:13-= 34:16:16:34.
IO2 is different from that formed by free
radical oxidation (20,22). Oxidation of methyl
linoleate with 10 2 produces the 9-, 10-, 12-and
13-monohydroperoxide isomers by attack of
102 at both ends of A9 and 12 double bonds
(20-23). On the other hand, free radical oxida-
t i o n of methyl linoleate yields only the 9- and
13-isomers (7,9). The isomeric distribution of
the hydroperoxylinoleoyl group of di-18:2
PC shown in Figure 6 agrees with that obtained

DISCUSSION
Interaction of 102 with olefins by an ene-
t y p e reaction results in aUylic hydroperoxides
(34). Unsaturated fatty acids (18-21) and
unsaturated triacylglycerols ~35) yield isomeric
monohydroperoxides when " 0 2 attacks their - - ~ - J % - t o t a l ion
double bonds. Thus, oxidation of unsaturated
phospholipids with 102 seems capable of
forming hydroperoxides by an ene-type reac-
tion. Soybean PC and di-18:2 PC were found to J~ mlz isomer
react with 102 at the position of unsaturated 173*315 13
fatty acid component to produce PC-MHP
during photosensitized oxidation. It is also J~" 187t301 12
probable that the oxidation of 1,2-diunsatu- 201t287 11
rated acyl PC with 102 yields PC-DHP by
215r 10
incorporating two oxygen molecules into each
unsaturated fatty acid component. Soybean 229 r 259 9
PC-DHP seems to be composed of 1,2-diun- I I J I I I
saturated molecular species, such as 18: l- 18: 2, Scon n u m b e r0
di-18:2 PC.
It has been found that the distribution of FIG. 6. Mass chromatography of isomeric methyl
the positional isomers of unsaturated fatty acid hydroxy octadecanoate obtained from hydroperoxy-
monohydroperoxides formed by oxidation with linoleate component of di-18:2 PC-MHP.

LIPID& VOL. 16, NO. 6 (1981)

432 J. TERAO, Y. HIROTA, M. KAWAKATSU AND S. MATSUSHITA

f r o m oxidatiGn o f m e t h y l linoleate w i t h 10 2. It B.P.S. Khambay, R.F. Garwood and G.C.L.

is t h e r e f o r e c o n c l u d e d t h a t the u n s a t u r a t e d acyl Weedon, Lipids 12:901 (1977).
9. Frankei, E.N., W.E. Neff, W.K. Rohwedder,
group o f PC is s u b j e c t t o t h e a t t a c k o f 102 in B.P.S. Khambay, R.F. Garwood and B.C.L.
the same m a n n e r as the c o r r e s p o n d i n g f a t t y Weedon, Lipids 12:908 (1977).
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fore, analysis o f t h e i s o m e r i c h y d r o p e r o x y f a t t y 15:163 (1980).
13. Nakano, M., T. Noguchi, K. Sugiota, H. Fuku-
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LIPIDS, VOL. 16, NO. 6 (1981)