Cis-trans isomerization of polyunsaturated fatty acid residues in phospholipids catalyzed by thiyl radicals

Article abstract of DOI:10.1021/ja0040969

Phospholipids containing trans-unsaturated fatty acid residues are the major products of the thiyl radical attack on L-α-phosphatidylcholine from soybean lecithin in homogeneous solution or in liposomes (LUVET). Thiyl radicals act as the catalyst for the cis-trans isomerization, and the number of catalytic cycles depends on the reaction conditions. The presence of ～0.2 mM oxygen does not influence the reaction outcome but accelerates the efficiency of cis-trans isomerization in homogeneous solution. Under these conditions, the PUFA peroxidation is found to be unimportant. A detailed study of the isomerization of methyl linoleate including product studies indicates the formation of a small amount of conjugated dienes that act as inhibitors. Indeed, all-trans-retinol substantially retarded the isomerization process.

Full text of DOI:10.1021/ja0040969

J. Am. Chem. Soc. 2001, 123, 4459-4468
4459
Cis-Trans Isomerization of Polyunsaturated Fatty Acid Residues in
Phospholipids Catalyzed by Thiyl Radicals
Carla Ferreri,†,‡ Cristina Costantino, Laura Perrotta, Laura Landi,§
†
†,§
|
,†
Quinto G. Mulazzani, and Chryssostomos Chatgilialoglu*
Contribution from the I.Co.C.E.A. and F.R.A.E., Consiglio Nazionale delle Ricerche, Via P. Gobetti 101,
4
0129 Bologna, Italy, Dipartimento di Chimica Biologica, UniVersit a` di Napoli “Federico II”,
Via Mezzocannone 16, 80134 Napoli, Italy, and Dipartimento di Biochimica “G. Moruzzi”,
UniVersit a` di Bologna, Via Irnerio 48, 40126 Bologna, Italy
ReceiVed NoVember 27, 2000
Abstract: Phospholipids containing trans-unsaturated fatty acid residues are the major products of the thiyl
radical attack on L-R-phosphatidylcholine from soybean lecithin in homogeneous solution or in liposomes
(LUVET). Thiyl radicals act as the catalyst for the cis-trans isomerization, and the number of catalytic cycles
depends on the reaction conditions. The presence of ∼0.2 mM oxygen does not influence the reaction outcome
but accelerates the efficiency of cis-trans isomerization in homogeneous solution. Under these conditions,
the PUFA peroxidation is found to be unimportant. A detailed study of the isomerization of methyl linoleate
including product studies indicates the formation of a small amount of conjugated dienes that act as inhibitors.
Indeed, all-trans-retinol substantially retarded the isomerization process.
Introduction
fish oils by heat, deodorization, and catalytic hydrogenation
7
processes; mono-trans-linoleic and R-linolenic acids are the
Unsaturated fatty acids present in the cell membranes play
an essential role in adaptation responses, which are correlated
to both the geometry and the number of the double bonds. Some
bacteria enzymatically convert unsaturated lipids from cis to
trans isomers without a shift of the double bond, as a strategy
for protecting themselves from increases in the ambient tem-
most commonly found, di-trans isomers being in minor quanti-
8
ties. The effect of trans-unsaturated fatty acids supplied by the
diet has recently raised some concern in human health and
nutrition since they have been correlated with higher serum
lipoprotein cholesterol and triglyceride levels and an increasing
9
risk of heart diseases. It is known that the various trans-fatty
perature or from the presence of high concentrations of toxic
substances.1,2 The number of double bonds has recently been
acids are inhibitors of the desaturation and chain-elongation
1
1,16
3
enzymes.
Linolelaidic acid can also interfere with the
correlated to thermotolerance in plants as well as to salt stress
tolerance in bacteria. The regulation of the fatty acid unsat-
urations affects the physical properties of the membrane bilayer
4
metabolism of linoleic acid and the biosynthesis of prostaglan-
1
7,18
dins.
However, the relationship between trans-fatty acids
(5) For reviews, see: (a) Wolff, R. L.; Precht, D.; Molkentin P. In Trans
(e.g., microviscosity and relative motional freedom of the lipid
Fatty Acids in Human Nutrition; S e´ b e´ dio, J.-L., Christie, W. W., Eds.; The
Oily Press: Dundee, Scotland, 1998; Chapter 1; pp 1-33. (b) Katan, M.
B.; Zock, P. L.; Mensink, R. P. Annu. ReV. Nutr. 1995, 15, 473-493.
molecules), and a fine-tuning is needed in order to acclimate
these organisms to changes of environmental conditions.
Enzymatic pathways for the cis-trans isomerization of
unsaturated fatty acid residues in mammals are unknown.
However, trans isomers can arise in cell membranes from
(
6) Mackie, R. I.; White, B. A.; Bryant, M. P. CRC Crit. ReV. Microbiol.
1
991, 17, 449-479.
(7) Ackman, R. G.; Hooper, S. N.; Hooper, D. L. J. Am. Oil Chem. Soc.
1974, 51, 42-49.
exogenous supplies as the food intake. Meat and milk contain
(8) In these industrial processes, both configurational and positional
isomers can be obtained. Therefore, the analytical conditions used for such
components are of crucial importance in order to separate and correctly
identify them. For example, see: (a) Kramer, J. K. G.; Fellner, V.; Dugan,
M. E. R.; Sauer, F. D.; Mossoba, M. M.; Yurawecz, M. P. Lipids 1997, 32,
5
∼
2-8% trans-fatty acid residues due to the biohydrogenation
of lipids by bacteria occurring in the first stomach of ruminant
6
animals. trans-Fatty acids are produced in much higher
1
219-1228. (b) Lie Ken Jie, M. S. F.; Mustafa, J. Lipids 1997, 32, 1019-
proportion (∼25-45%) in the manufacturing of vegetable and
1034. (c) Ratnayake, W. N. M. In Trans Fatty Acids in Human Nutrition;
*
Corresponding author. Phone: 39-051-6398309. Fax: 39-051-6398349.
S e´ b e´ dio, J.-L., Christie, W. W., Eds.; The Oily Press: Dundee, Scotland,
E-mail: chrys@area.bo.cnr.it.
1998; Chapter 4; pp 115-161.
†
10
I.Co.C. E. A.
Universit a` di Napoli “Federico II”.
Universit a` di Bologna.
F. R. A. E.
(9) In a series of studies performed in several countries the presence
‡
11
12, 13
14
of trans isomers in human milk, blood,
found to be correlated with dietary intake. In this respect, a significant
difference among countries has been pointed out, ranging from values of
12-20% in North America to 1-2% in Mediterranean countries.
(10) van Poppel, G. Lancet 1998, 351, 1099.
(11) Carlson, S. E.; Clandinin, M. T.; Cook, H. W.; Emken, E. A.; Filer,
L. T., Jr. Am. J. Clin. Nutr. (Suppl.) 1997, 66, 715S-736S.
(12) Siguel, E. N.; Lerman, R. Am. J. Cardiol. 1993, 71, 916-920.
Willett, W.; Stampfer, M. J.; Manson, J. E.; Colditz, G. A.; Speizer, F. E.;
Rosner, B. A.; Sampson, L. A.; Hennekens, C. H. Lancet 1993, 341, 581-
585.
and adipose tissues has been
§
|
1
5
(
1) For a review, see: Keweloh, H.; Heipieper, H. J. Lipids 1996, 31,
1
29-137.
(2) For some recent work, see: Loffeld, B.; Keweloh, H. Lipids 1996,
3
1, 811-815. Holtwick, R.; Meinhardt, F.; Keweloh, H. Appl. EnViron.
Microbiol. 1997, 63, 4292-4297. Okuyama, H.; Ueno, A.; Enari, D.; Morita,
N.; Kusano, T. Arch. Microbiol. 1998, 169, 29-35.
(3) Murakami, Y.; Tsuyama, M.; Kobayashi, Y.; Kodama, H.; Iba, K.
Science 2000, 287, 476-479.
4) Allakhverdiev, S. I.; Nishiyama, Y.; Suzuki, I.; Tasaka, Y.; Murata,
N. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5862-5867.
(
(13) Hodgson, J. M.; Walqvist, M. L.; Boxall, J. A.; Balazs, N. D.
Atherosclerosis 1996, 120, 147-154.
1
0.1021/ja0040969 CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/19/2001

4460 J. Am. Chem. Soc., Vol. 123, No. 19, 2001
Ferreri et al.
Scheme 1
Scheme 2
and pathological conditions is far to be understood, and up to
now, the presence and effects of trans-fatty acid residues in
2
1
mammal cells have only been correlated to the dietary intake.
process. Thiyl radicals can attack the double bond of the
membrane lipids randomly causing a change in the configuration
of the hydrocarbon tails.
Radical-based damage of biologically relevant molecules has
increasingly attracted the interest of researchers from different
scientific fields, from chemistry to medicine. Radical processes
involving membrane phospholipids are mainly referring to
We suggested that the geometrical isomerization of unsatur-
ated lipids could be an endogenous process. The biological
consequences are predictable as they are similar to the alterations
already observed when trans-unsaturated fatty acids taken from
22
polyunsaturated fatty acid (PUFA) peroxidation. More recently
the homolytical cleavage of lysophospholipids23 and the cis-
trans isomerization of unsaturated fatty acid residues have been
9
2
4-26
the diet are included into membranes. Therefore, we pursued
reported.
our interest toward polyunsaturated lipids since the event of an
endogenous process could bring about an even more important
biological effect. In fact, since the trans lipid geometry resembles
We have shown that phospholipids containing trans-unsatur-
ated fatty acids are the only products of the thiyl radical attack
on natural dioleoylphosphatidylcholine.24 The mechanism that
2
8
that of saturated lipids, the geometrical isomerization can be
considered equivalent to a decrease of the number of unsatura-
tions in the cell membrane, thus causing an impairment of the
we conceived for this transformation includes the addition of
thiyl radicals to the cis double bond of an oleate moiety to give
the intermediate radical 1a, half-rotation about the carbon-
3
cell adaptation systems.
carbon bond to give 1b, and ejection of the thiyl radical by
â-scission (cf. Scheme 1).27 We have also modeled the occur-
Sch o¨ neich et al. studied the reaction of polyunsaturated fatty
acids with a variety of thiyl radicals by pulse radiolysis
rence of such a reaction in cell membranes using lipid vesicles,
29
naturally occurring thiols, and aerobic conditions,24 showing that
technique. A variety of kinetic information was obtained based
on the buildup of pentadienyl-type radical 2 formed according
to Scheme 2. For example, rate constants in the range (0.6-
the isomerization in lipid bilayer aggregates is still an effective
(14) Aro, A.; Kardinaal, A. F. M.; Salminen, I.; Kark, J. D.; Riemerama,
7
-1 -1
3
.1) × 10 M
s
are obtained for the reactions of biologically
R. A.; Delgado-Rodriguez, M.; Gomez-Aracena, J.; Huttunen, J. K.;
Kohlmeler, L.; Martin, B. C.; Martin-Moreno, J. M.; Mazaev, V. P.;
Ringstad, J.; Thamm, M.; van’t Veet, P.; Kok, F. J. Lancet 1995, 345, 273-
relevant thiyl radicals with linoleic acid. On the basis of several
assumptions, the authors proposed that ∼50% of the thiyl
radicals abstracts the bisallylic hydrogen whereas the remaining
2
78.
(
15) Craig-Schmidt, M. C. In Trans Fatty Acids in Human Nutrition;
S e´ b e´ dio, J.-L., Christie, W. W., Eds.; The Oily Press: Dundee, Scotland,
•
RS add to the double bond to form the radical adduct 3. In the
absence of oxygen, the latter pathway is reversible, whereas in
the presence of oxygen, both radicals 2 and 3 react to form the
corresponding peroxyl radicals (Scheme 2). However, as the
authors themselves pointed out, product studies are necessary
in order to address more properly the reaction mechanism.29
Schwinn et al. reported the thiyl radical-induced cis-trans
1
2
1
998; Chapter 3; pp 57-113.
(
16) For a review, see: Koletzko, B.; Decsi, T. Clin. Nutr. 1997, 16,
29-237.
(17) Kinsella, J. E.; Bruckner, G.; Mai, J.; Shimp, J. Am. J. Clin. Nutr.
981, 34, 2307-2318.
(18) It is worth pointing out that a few data about the fate of trans-
polyenes have been reported so far, and most of the studies have been carried
out in vivo and in vitro using only animals as models. Studies on mice
using isotopically labeled 18:2 isomers showed that the metabolites are
incorporated into liver, plasma, heart, and brain lipids.
19) S e´ b e´ dio, J.-L.; Chardigny, J.-M. In Trans Fatty Acids in Human
Nutrition; S e´ b e´ dio, J.-L., Christie, W. W., Eds.; The Oily Press: Dundee,
Scotland, 1998; Chapter 6; pp 191-215.
1
9
25
isomerization of methyl linoleate in methanol. On the basis
2
9
2
0
of the previous mechanistic considerations, they postulated
that the trans isomers of linoleate moieties represent the fate of
the pentadienyl radical, which reacts with thiols in different
(
2
5a
25b
conformations. The same group later revised their conclusions
(20) Beyers, E. C.; Emken, E. A. Biochim. Biophys. Acta 1991, 1082,
24
2
75-284.
and, on the basis of our preliminary communication, suggested
a mechanistic scheme for the cis-trans isomerization of methyl
linoleate similar to that reported in Scheme 1. Balazy and co-
(
21) Ip, C.; Marshall. J. R. Nutr. ReV. 1996, 54, 138-145.
(22) For some representative reviews, see: Porter, N. A. Acc. Chem.
Res. 1986, 19, 262-268. Niki, E. In Organic Peroxides; Ando, W., Ed.;
Wiley: New York; 1992; pp 764-787. Barclay, L. C. R. Can. J. Chem.
•
workers have shown that the reaction of NO2 radicals with
1
993, 33, 1-16.
arachidonic acid produces a mixture of isomers, having one trans
(
23) Muller, S. N.; Batra, R.; Senn, M.; Giese, B.; Kisel M.; Shadyro,
O. J. Am. Chem. Soc. 1997, 119, 2795-2803.
24) Chatgilialoglu, C.; Ferreri, C.; Mulazzani, Q. G.; Ballestri, M.; Landi,
(28) The similarity between a mono-trans and a saturated fatty acid was
confirmed by steady-state fluorescence polarization studies of vesicles
composed of phospholipids from the liver mitochondria of rats fed with
elaidic acid. See: Wolff, R. L.; Entressangles, B. Biochim. Biophys. Acta
1994, 1211, 198-206.
(29) Sch o¨ neich, C.; Dillinger, U.; von Bruchhausen, F.; Asmus, K.-D.
Arch. Biochem. Biophys. 1992, 292, 456-467. Sch o¨ neich, C.; Bonifacic,
M.; Dillinger, U.; Asmus, K.-D. In Sulfur-Centered ReactiVe Intermediates
in Chemistry and Biology; Chatgilialoglu, C., Asmus, K.-D., Eds.; Plenum
Press: New York, 1990; pp 367-376. Sch o¨ neich, C.; Asmus, K.-D.;
Dillinger, U.; von Bruchhausen, F. Biochem. Biophys. Res. Commun. 1989,
161, 193-120.
(
L. J. Am. Chem. Soc. 2000, 122, 4593-4601. Ferreri, C.; Costantino, C.;
Landi, L.; Mulazzani, Q. G.; Chatgilialoglu, C. Chem. Commun. 1999, 407-
4
08.
(25) (a) Schwinn, J.; Sprinz, H.; Dr o¨ ssler, K.; Leistner, S.; Brede, O.
Int. J. Radiat. Biol. 1998, 74, 359-365. (b) Sprinz, H.; Schwinn, J.; Naumov,
S.; Brede, O. Biochem. Biophys. Acta 2000, 1483, 91-100.
(26) Jiang, H.; Kruger, N.; Lahiri, D. R.; Wang, D.; Vat e` le, J.-M.; Balazy,
M. J. Biol. Chem. 1999, 274, 16235-16241.
27) Chatgilialoglu, C.; Ballestri, M.; Ferreri, C.; Vecchi, D. J. Org.
Chem. 1995, 60, 3826-3831.
(

Cis-Trans Isomerization of PUFA by Thiyl Radicals
J. Am. Chem. Soc., Vol. 123, No. 19, 2001 4461
and three cis double bonds.26 They proposed a reversible
•
addition of NO2 radicals to arachidonic moieties analogous to
the mechanism shown in Scheme 1.
We report herein the reaction of thiyl radicals with mono-
and polyunsaturated phospholipids in the naturally occurring
mixture of purified L-R-phosphatidylcholine from soybean
lecithin. The geometrical isomerization has been studied both
in solution and in lipid vesicles (LUVET), which model the
cell membrane. The isomerization of methyl linoleate has been
revisited, and product studies allow proposing a detailed
mechanistic scheme. The effectiveness of the cis-trans isomer-
ization under aerobic conditions and the inhibition of this chain
process by conjugated dienes will also be addressed in some
details.
Figure 1. (a) Time profiles of methyl linoleate (0.15 M) isomerization
with 75 mM PhSH and 30 mM AMVN in t-BuOH at 54 °C: (b) methyl
linoleate; (2) mono-trans isomers; (9) methyl linolelaidate. (b) Time
profiles of methyl linolelaidate isomerization (0.15 M) with 75 mM
PhSH and 30 mM AMVN in t-BuOH at 54 °C: (9) methyl
linolelaidate; (2) mono-trans isomers; (b) methyl linoleate.
Results and Discussion
Generation of Radicals in t-BuOH. Thiyl radicals were
produced by the reaction of an alkyl radical with benzenethiol
(PhSH) or â-mercaptoethanol (HOCH2CH2SH). Alkyl radicals
were generated by either thermal decomposition of azobis-
Isomerization of Methyl Linoleate.36 A solution of 0.15 M
methyl linoleate (9cis,12cis-18:2), 0.075 M PhSH, and 0.03 M
AMVN in t-BuOH was heated at 54 °C for 7 h. The reaction
was monitored by GC (Rtx-2330 column), and its time profile
is shown in Figure 1a. The disappearance of the 9cis,12cis-
18:2 (solid circles) was replaced by the formation of mono-
trans isomers (solid triangles) and 9trans,12trans-18:2 (solid
squares). The two mono-trans isomers, i.e., 9trans,12cis-18:2
and 9cis,12trans-18:2, were found in equal amounts and are
reported together. The overall yield of the four isomers decreases
linearly with the time and is found to be 85% after 7 h by using
methyl palmitate as internal standard. Furthermore, 6% of four
conjugated dienes (i.e., cis- and trans-9,11- and -10,12-
octadecadienoic acid methyl esters) were singled out, based on
the comparison with a commercially available mixture. A
parallel GC analysis by using a HP-5 column also showed a
broad peak (or cluster of peaks) that was assigned by GC/MS
to PhSH adducts of methyl linoleate. Chromatographic attempts
to isolate this fraction from the other components failed. Figure
1b shows the analogous reaction in which the starting material
is methyl linolelaidate. The disappearance of the 9trans,12trans-
18:2 (solid squares) was replaced by the formation of mono-
trans isomers (solid triangles) whereas the formation of methyl
linoleate (solid circles) is close to zero. In agreement with the
preceding experiment, after 7 h the yields were found to be 84
and 6% for the geometrical isomers and the conjugated dienes,
respectively.
(
(
isobutyronitrile) (AIBN) and azobis(dimethylvaleronitrile)
AMVN) at 71 and 54 °C, respectively (eq 1),30 or γ-irradiations
of N2O-saturated t-BuOH.
∆
•
RsNdNsR f N + 2R
(1)
2
Radiolysis of t-BuOH leads to the species esol- and
CH2C(CH3)2OH as shown in eq 2. In N2O-saturated solutions,
•
esol- is transformed into the HO radical (eq 3). Hydrogen
•
•
•
abstraction from t-BuOH by HO produces CH2C(CH3)2OH (eq
4
8
-1 -1
, k4 ) 6.0 × 10 M s ), and therefore, we can consider the
• -1 31
G value of CH2C(CH3)2OH radical to be ∼0.65 µmol J .
•
The CH2C(CH3)2OH in turn reacts with the HOCH2CH2SH to
7
-1
give the corresponding thiyl radical (eq 5, k5 = 5 × 10 M
-
1 33
s ).
+
Η
esol- + N O f N + HO
•
(3)
2
2
•
•
HO + (CH ) COH f CH C(CH ) OH + H O (4)
3
3
2
3 2
2
•
•
CH C(CH ) OH + RSH f (CH ) COH + RS
(5)
To reach the equilibrium mixture, methyl linoleate or methyl
linolelaidate or a commercially available mixture of the four
geometrical isomers 18:2 (9cis,12cis/9cis,12trans/9trans,12cis/
2
3 2
3 3
(30) At these temperatures, the half-life times are longer than 4 h. Taken
from the half-life time vs temperature curves reported by Akzo Chemie.
9
trans,12trans ) 8/20/20/52) was allowed to react at 71 °C
-
1
•
(
31) Based on the total G ) 0.61 µmol J in water (eq 6) and G( CH2-
with an excess of PhSH (0.45 M) and AIBN as the initiator.
The final isomeric composition is independent of the starting
material and corresponds to 9cis,12cis/9cis,12trans/9trans,12cis/
-1 32 •
OH) ) 0.67 µmol J in methanol, the assumption of G[ CH2C(CH3)2-
-
1
OH] ) 0.65 µmol J seems reasonable.
32) Spinks, J. W. T.; Woods, R. J. An Introduction to Radiation
Chemistry, 3rd ed.; Wiley: New York 1990; p 421.
(
9
trans,12trans ) 4/16/16/64. By using the Boltzmann distribu-
3
4
•
(
33) It is known that in water the CH2C(CH3)2OH abstracts hydrogen
-
1
7
-1 -1
tion expression, we obtained ∆E values of 0.87 kcal mol
from the HOCH2CH2SH with a rate constant of k5 ) 8.2 × 10 M
s .
The rates of thiol trapping of alkyl radicals are solvent dependent.35 The
rate constant for the reaction of a primary alkyl radical with octanethiol is
between methyl linoleate and the mono-trans isomers and
between the mono-trans isomers and methyl linolelaidate. These
values account for the difference in the stability of the four
isomers. Indeed, for cis- and trans-2-butene, ∆fH°(trans) -
7
-1 -1
35
measured to be 1.9 × 10 M
s
at 30 °C in THF.
(
34) (a) Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross, A. B.
J. Phys. Chem. Ref. Data, 1988, 17, 513 and references therein. (b) Ross,
A. B., Mallard, W. G.; Helman, W. P.; Buxton, G. V.; Huie, R. E.; Neta,
P. NDRL-NIST Solution Kinetic Database-Ver. 3, Notre Dame Radiation
Laboratory, Notre Dame, IN and NIST Standard Reference Data, Gaith-
ersburg, MD, 1998.
-
1 37
∆fH°(cis) ) -1.0 kcal mol .
(36) The following nomenclature system will be used throughout the
article: methyl linoleate or 9cis,12cis-18:2, methyl linolelaidate or 9trans,-
12trans-18:2, and their mono-trans isomers, i.e., 9trans,12cis-18:2- and 9cis,-
12trans-18:2.
(35) Tronche, C.; Martinez, F. N.; Horner, J. H.; Newcomb, M.; Senn,
M.; Giese, B. Tetrahedron Lett. 1996, 33, 5845-5848.

4462 J. Am. Chem. Soc., Vol. 123, No. 19, 2001
Ferreri et al.
Figure 3. (a) 9cis,12cis-18:2 (b, O), 9trans,12cis-18:2, and 9cis,-
Figure 2. Time profiles of methyl linoleate (b) or methyl oleate (O)
in t-BuOH at 54 °C. (a) Isomerization of methyl linoleate (0.15 M)
1
2trans-18:2 (2, 4) or 9trans,12trans-18:2 (9, 0) vs dose from the
-1
γ-radiolysis (23.3 Gy min ) of methyl linoleate with 75 mM
with 75 mM HOCH
M) with 75 mM HOCH
of a mixture of methyl linoleate (0.15 M) and methyl oleate (0.15 M)
2 2
CH SH and 30 mM AMVN or methyl oleate (0.15
HOCH
2
CH
2
SH in N
2
O-saturated t-BuOH at 22 °C: (b, 2, 9) without
2
CH SH and 30 mM AMVN. (b) Isomerization
2
-4
oxygen; (O, 4, 0) with 2.34 × 10 M oxygen. (b) G[-(9cis,12cis-
1
8:2)] vs dose (b) without oxygen and (O) with oxygen.
2 2
with 75 mM HOCH CH SH and 30 mM AMVN.
Figure 2a also shows the time profile of the disappearance of
methyl oleate under identical condition (open circles) taken from
our previous work. It is evident that the isomerization of
methyl linoleate is much slower than the corresponding reaction
of methyl oleate. To throw some light on this unexpected result,
we studied the isomerization of a mixture of methyl linoleate
Scheme 3
2
4
(0.15 M) and methyl oleate (0.15 M) with HOCH2CH2SH (0.075
M) in t-BuOH using AMVN (0.03 M) at 54 °C. Figure 2b shows
this experiment. The efficiency of methyl linoleate isomerization
is the same in the presence or absence of methyl oleate, whereas
the disappearance of methyl oleate is dramatically decreased
(cf. Figure 2a and b). Furthermore, in Figure 2b, it can be seen
that the methyl linoleate isomerized 2 times faster than methyl
oleate; that is, the three double bonds (two of linoleate and one
of oleate) isomerize with the same efficiency. The above
observation suggests that, during the methyl linoleate isomer-
ization, an inhibitor is forming that retards the isomerization.
Figure 3a shows the irradiation dose profiles of the dis-
appearance of 9cis,12cis-18:2 (solid circles), the formation of
the two mono-trans isomers (solid triangles), and 9trans,12trans-
18:2 (solid squares). It is worth pointing out that the data are a
combination of two independent experiments, and therefore, the
reproducibility is quite good. Also in the irradiation experiments,
the four geometrical isomers account for at least 95% of the
yield without any evidence for the formation of conjugated
These experiments show that the initial disappearance of
methyl linoleate is replaced by the two mono-trans isomers,
which in their turn are the precursors of the di-trans isomer,
i.e., a step-by-step isomerization (Scheme 3).38a The formation
of conjugated dienes is probably due to the abstraction of
•
bisallylic hydrogen by the PhS radical to give the pentadienyl
radical 2 (Scheme 2) followed by hydrogen abstraction from
PhSH.3
8b
Figure 2a (solid circles) shows the disappearance of methyl
linoleate when the above azo-initiated reaction was run in the
presence of HOCH2CH2SH (0.075 M). In comparison with the
PhSH experiment (Figure 1a, solid circles), the reaction is
considerably slower in accordance with the relative reactivities
-
1
dienes. The disappearance of the starting material (mol kg )
-
1
divided by the absorbed dose (1Gy ) 1 J kg ) gives the
radiation chemical yield or G[-(9cis,12cis-18:2)]. Figure 3b
(solid circles) shows the plot of G[-(9cis,12cis-18:2)] versus
•
•
24,40
of PhS and HOCH2CH2S radicals.
However, under these
conditions, the yield of the four geometrical isomers is 93%
whereas the four conjugated dienes (i.e., cis- and trans-9,11-
and -10,12-octadecadienoic acid methyl esters) are absent.
-
1
dose. The extrapolation to zero dose gives G = 9 µmol J .
•
-1
31
Assuming that the G(RS ) is 0.65 µmol J (eqs 4 and 5), we
calculated the catalytic cycle to be 14 at the initial phase. For
the analogous isomerization of methyl oleate, a catalytic cycle
(
37) Handbook of Chemistry and Physics, 77th ed.; Lide, D. R., Ed.;
CRC Press: Boca Raton, FL, 1996-97.
38) (a) The kinetics and equilibrium constant for the reaction PhS +
2
4,41
•
of 350 was calculated.
(
•
3
-1 -1
CH2dCHR h PhSCH2C( )HR, are reported to be kf ) 4.0 × 10 M
s
,
Figure 3a (O, 4, 0) also shows the dose profile of the
7
-1
-1
kr ) 3.3 × 10 s , and K ) 0.0012 M , respectively. See: Ito, O. In
S-Centered Radicals; Alfassi, Z. B., Ed.; Wiley: Chichester; U.K., 1999;
pp 193-224. (b) The bond dissociation energies of (CH2dCH)2CHsH and
isomerization from a solution saturated with a mixture of N2O/
42
air containing 10% oxygen. Again, the data are a combination
of two independent experiments indicating a high reproduc-
-
1
39,40a
PhSsH are 76.6 and 80.8 kcal mol , respectively.
Therefore, the
-
1
formation of pentadienyl radical is ∼4 kcal mol exothermic.
(
39) Clark, K. B.; Culshaw, P. N.; Griller, D.; Lossing, F. P.; Martino
(41) The terms “catalytic cycle” and “thiyl radical-catalyzed reaction”
are more appropriate that “chain length” and “radical chain reaction”, which
have been used in our previous work.24
Sim o˜ es, J. A.; Walton, J. C. J. Org. Chem. 1991, 56, 5535-5539. Fort, R.
C., Jr.; Hrovat, D. A.; Borden, W. T. J. Org. Chem. 1993, 58, 211-216.
(40) For example, see: (a) Chatgilialoglu, C.; Guerra, M. In Supplement
(42) The solubility of molecular oxygen in t-BuOH is determined to be
-
3
43
S: The Chemistry of Sulfur-containing Functional Groups; Patai, S.,
Rappoport, Z., Eds.; Wiley: London; 1993; Chapter 8; pp 363-394. (b)
Chatgilialoglu, C.; Bertrand, M. P.; Ferreri, C. In S-Centered Radicals;
Alfassi, Z. B., Ed.; Wiley: Chichester, U.K., 1999; pp 312-354.
2.34 × 10 M at room temperature. Therefore, 10% oxygen is equal to
-4
2.34 × 10 M.
(43) Cipollone, M.; di Palma, C.; Pedulli, G. F. Appl. Magn. Reson. 1992,
3, 98-102.

Cis-Trans Isomerization of PUFA by Thiyl Radicals
J. Am. Chem. Soc., Vol. 123, No. 19, 2001 4463
Scheme 4
ibility. In comparison with the deoxygenated solution experiment
(
b, 2, 9), the reaction is substantially accelerated. Indeed, the
Figure 4. Time profiles of methyl linolenate (0.15 M) isomerization
disappearance of 9cis,12cis-18:2 (open circles) and the appear-
ance of the two mono-trans isomers (open triangles) and di-
trans isomer (open squares) is faster at the same dose. Figure
with 75 mM HOCH
2 2
CH SH in t-BuOH: (b) methyl linolenate; (2)
mono-trans isomers; (1) di-trans isomers; (9) all-trans isomer. (a) 30
-
1
mM AMVN at 54 °C. (b) From the γ-radiolysis (24.9 Gy min ) at 22
C.
3
b (open circles) shows the G[-(9cis,12cis-18:2)] versus dose
°
-
1
which gives a G = 19 µmol J by extrapolation to zero dose
and a catalytic cycle of 29 at the initial phase. The increase of
prefer to combine rather than abstract hydrogen from the thiol
-
4
catalytic cycle from 14 to 29 in the presence of 2.34 × 10
M
46
under our experimental conditions. We suggest that the
oxygen42 is not easily explained. Indeed, this behavior is
opposite to the one observed for the isomerization of methyl
oleate, in which the catalytic cycle was decreasing from 350 to
formation of the dimers 4 (containing two moieties of conjugated
dienes) during the isomerization process is responsible for the
observed inhibition. In the presence of the molecular oxygen,
the substituted pentadienyl radical should give the corresponding
peroxyl radicals that lead to final products with lower inhibiting
properties than the dimers 4.
2
4,41
1
30 in the presence of the same amount of oxygen.
To investigate further the dichotomy between the isomeriza-
tion of methyl oleate and linoleate, we have undertaken a careful
examination of the products formed in the following reaction.
A 0.15 M solution of methyl linoleate (1.5 mmol) and HOCH2-
CH2SH (0.075 M) in t-BuOH was irradiated for 7 h. Flash
chromatography of the reaction mixture using n-hexane as the
eluent gave a first fraction of the geometrical isomers in 93%
yield. Further elution with 8/2 n-hexane/diethyl ether gave two
other fractions corresponding to a mixture of dimeric conjugated
dienes of type 4 (4% yield; Rf ) 0.47) and to a mixture of thiol-
Isomerization of Methyl Linolenate. For comparison with
methyl linoleate, we studied the isomerization of 0.15 M methyl
linolenate (9cis,12cis,15cis-18:3) with HOCH2CH2SH (0.075 M)
in t-BuOH using either AMVN (0.03 M) at 54 °C or γ-irradia-
tion of a N2O-saturated solution at 22 °C. Aliquots of the
solution were withdrawn at different times and examined by
GC analysis. Similar results were obtained under the two
experimental conditions. Figure 4 (solid circles) shows the time
profile of the disappearance of methyl linolenate, the formation
of the three mono-trans isomers (solid triangles up), the three
di-trans isomers (solid triangles down), and the all-trans isomer
4
7a,b
(solid squares).
It is worth pointing out that after 7 h of
irradiation a 92% yield of the eight isomers was obtained. A
TLC examination of the reaction mixture evidenced the forma-
tion of two secondary products, which, in analogy with the
byproducts observed in the linoleate reaction, were attributed
fatty acid adducts of type 5 (2% yield; Rf ) 0.16), respectively.
The mixture of dimeric conjugated dienes of type 4 suggests a
4
4
to dimers 4 and adducts 5 (R ) 0.57 and 0.11, respectively, in
coupling of the pentadienyl radicals at positions 9 and 13. On
the other hand, the adducts of type 545 correspond to the initial
addition of thiyl radicals to positions 10 and 12 followed by
hydrogen abstraction from the thiol prior to â-elimination of
f
7/3 n-hexane/diethyl ether).
The plot of G[-(9cis,12cis,15cis-18:3)] versus dose obtained
-
1
from Figure 4b gives a G = 8 µmol J by extrapolation to
zero dose and a catalytic cycle of 12 at the initial phase. Indeed,
this behavior is similar to the one observed for the isomerization
of methyl linoleate and we suggest that the mechanism reported
in Scheme 4 also operates in this case.
4
0
thiyl radicals.
Scheme 4 describes the reaction of methyl linoleate with
•
HOCH2CH2S radical in the absence or presence of molecular
oxygen. The main path is the formation of the geometrical
isomers by the mechanism described in Scheme 3. A minor
path is the hydrogen abstraction from the bisallylic position to
Isomerization of L-r-Phosphatidylcholine (PC) from Soy-
bean Lecithin in t-BuOH. A set of experiments similar to those
46
give a delocalized pentadienyl radical. The absence of octa-
decanoic conjugated dienes indicates that pentadienyl radicals
-
1 40
(
46) The bond dissociation energy of RS-H is 88.6 kcal mol
.
-1
Therefore, the formation of pentadienyl radical is ∼12 kcal mol
exothermic whereas the reaction of pentadienyl radical with RSH will be
-
1
(
44) Culshaw, P. N.; Walton, J. C.; Hughes, L.; Ingold, K. U. J. Chem.
Soc., Perkin Trans. 2 1993, 879-886.
45) This attribution is based on COSY, NOE, and DEPT NMR
∼8 kcal mol more endothermic than the corresponding reaction with
PhSH.38
(
(47) (a) The GC peaks of two of the di-trans isomers, i.e., 9cis,12trans,-
15trans-18:3 and 9trans,12cis,15trans-18:3, are overlapping under our best
analytical conditions (cf. Figure 5b). (b) Contrary to isomerization of methyl
linoleate in t-BuOH where the two mono-trans isomers were formed in
equal amounts, the three mono-trans isomers of methyl linolenate are formed
in different amounts. For example, the experiment in Figure 4b after 7 h
gave a ratio of 9.9/11.8/12.7 for 9cis,12cis,15trans-18:3, 9cis,12trans,15cis-
18:3, and 9trans,12cis,15cis-18:3, respectively.
experiments. The two protons attached to the double bond correlate with
three signals at 1.98, 2.12, and 2.23 ppm, which integrate for 2:1:1 protons,
respectively. The resonance at 1.98 ppm has been assigned to the allylic
hydrogens in C-8 or C-14. The signals at 2.12 and 2.23 ppm have been
assigned to the two diastereotopic allylic hydrogens in C-11. The DEPT
experiment evidenced the resonances of the tertiary carbon bearing the sulfur
substituent at 45.1 and 45.8 ppm, which refer to the two adducts.

4464 J. Am. Chem. Soc., Vol. 123, No. 19, 2001
Ferreri et al.
Figure 6. Fatty acid residues vs dose from the γ-radiolysis (23.3 Gy
-
1
min ) of PC from soybean lecithin with 75 mM HOCH
2 2
CH SH in
N
2
O-saturated t-BuOH at 22 °C: (b, 2, 9) without oxygen; (O, 4, 0)
-
4
with 2.34 × 10 M oxygen. (a) 9cis-18:1 (b, O) or 9trans-18:1(9,
). (b) 9cis,12cis-18:2 (b, O), 9cis,12trans-18:2, and 9trans,12cis-
8:2 (2, 4) or 9trans,12trans-18:2 (9, 0). (c) 9cis,12cis,15cis-18:3
b, O), mono-trans isomers (2, 4), or di-trans isomers (1, 3).
0
1
(
Figure 5. (a) GC chromatogram of fatty acid composition obtained
by transesterification of the commercially available L-R-phosphatidyl-
choline from soybean lecithin. (b) GC chromatogram of fatty acid
composition obtained after isomerization. Peaks: (1) 16:0, (2) 18:0,
(3) 9trans-18:1, (4) 11trans-18:1, (5) 9cis-18:1, (6) 11cis-18:1, (7)
Figure 7. G[-(9cis-18:1)] (b), G[-(9cis,12cis-18:2)] (2), or G[-(9cis,-
12cis,15cis-18:3)] (9) vs dose. (a) Without oxygen. (b) With 2.34 ×
9trans,12trans-18:2, (8) 9cis,12trans-18:2, (9) 9trans,12cis-18:2, (10)
9cis,12cis-18:2, (11) 9trans,12trans,15trans-18:3, (12+13) 9cis,12trans,-
15trans-18:3, and 9trans,12cis,15trans-18:3, (14) 9cis,12cis,15trans-
18:3, (15) 9trans,12trans,15cis-18:3, (16) 9cis,12trans,15cis-18:3, (17)
9trans,12cis,15cis-18:3, and (18) 9cis,12cis,15cis-18:3.
-
4
10 M oxygen.
geometrical isomers of 18:1, 18:2, and 18:3 components is
visible. The conversion of the initial material to the final mixture
can be considered quantitative since the sum of various
components accounts for a ∼95% yield.
with methyl linoleate or methyl linolenate was carried out with
a t-BuOH solution of PC since it is known that phospholipids
Figure 6a shows the dose profiles of methyl oleate consump-
tion (solid circles) and methyl elaidate formation (solid squares).
Figure 6b shows the time profiles of methyl linoleate consump-
tion (solid circles) and the formation of two mono-trans isomers
48
do not aggregate in this solvent. The initial fatty acid
composition of the commercially available L-R-phosphatidyl-
choline from soybean lecithin was obtained by transesterification
followed by GC analysis (Figure 5a) and found to be: methyl
palmitate (14.5%), methyl stearate (3.8%), methyl oleate
(solid triangles) and di-trans isomers (solid squares). Figure 6c
shows the time profiles of methyl linolenate consumption (solid
circles) and the formation of three mono-trans isomers (solid
triangles-up) and three di-trans isomers (solid triangles-down).
The all-trans isomer if present is lower than our GC instrument
sensitivity.
(
11.9%), methyl vaccenate (1.3%), methyl linoleate (63.5%),
and methyl linolenate (6.3%). A PC/chloroform solution (3 mL;
.15 mmol of fatty acid contents) was evaporated in a test tube
0
under an argon stream. The thin film formed was kept under
vacuum for 30 min and then a 1 mL t-BuOH was added. After
saturation of the solution with N2O, the HOCH2CH2SH (0.075
mmol) was added prior to γ-irradiation. Aliquots (100 µL) of
the reaction mixture were processed at different times. After
transesterification49 of the phospholipid, the various cis/trans
ratios were obtained by GC analysis. An example is given in
Figure 5b in which the excellent separation of all possible
Figure 7a shows the plot of G values versus dose for oleic
(
circles), linoleic (triangles), and linolenic acid (squares)
residues. The extrapolation to zero dose gives G values of 0.5,
-1
6
.4, and 1.1 µmol J , respectively, which correspond to a total
-1
•
-1
G ) 8 µmol J . Assuming that the G(RS ) is 0.65 µmol J
eqs 4 and 5), we calculated a catalytic cycle 12 at the initial
31
(
phase. We have also considered the catalytic cycle for each
component of the lipid mixture. The total molarity of CdC
double bonds, based on the PC fatty acid composition (7.6%
oleate, 80.6% linoleate, and 11.8% linolenate residues), can be
calculated to be 0.237 M. By dividing these percentages by the
corresponding G values (i.e., 7.6/0.5, 60.6/6.4, and 11.8/1.1),
(
48) Barclay, L. R. C.; McNeil, J. M.; VanKessel, J.; J. Forrest, B.; Porter,
N. A.; Lehman, L. S.; Smith, K. J.; Ellington, J. C., Jr. J. Am. Chem. Soc.
984, 106, 6740-6747.
49) The transesterification in alkaline medium is preferable, as reported
1
(
by: Kramer, J. F. K.; Fellner, V.; Dugan, M. E. R.; Sauer, F. D.; Mossoba
M. M.; Yurawecz, M. P. Lipids, 1997, 32, 1219-1228.

Cis-Trans Isomerization of PUFA by Thiyl Radicals
J. Am. Chem. Soc., Vol. 123, No. 19, 2001 4465
we have again obtained an average catalytic cycle of 12,
•
suggesting that at zero dose the attack of the RS radical to
double bonds is only a statistic.
Figure 6 (open symbols) shows the dose profile of the
isomerization from a solution saturated with a mixture of N2O/
air containing 10% oxygen.42 In comparison with the deoxy-
genated solution experiment (solid symbols), the reaction is
considerably faster in all cases. To quantify this increase, in
Figure 7b we plotted the G value versus dose for each of the
unsaturated fatty acid residues. The extrapolation to zero dose
gives G values of 0.9, 8.8, and 1.3 µmol J- for methyl oleate,
methyl linoleate, and methyl linolenate, respectively, which
1
-
1
corresponds to a total G ) 11 µmol J and to a catalytic cycle
of 17 at the initial phase. Therefore, in the presence of 2.34 ×
-
4
42
1
1
0
M oxygen, an increase of the catalytic cycle from 12 to
Figure 8. Irradiation (23.3 Gy min-1) of PC from soybean lecithin in
LUVET (0.15 M fatty acid contents) with 7 mM HOCH CH SH at 22
2 2
7 is observed.
It is gratifying to see that the isomerization behavior is similar
°C. (a) Unsaturated fatty acid residues vs dose: methyl linoleate (b),
mono-trans isomers (2), or methyl linolelaidate (9) without oxygen;
to the analogous reaction of methyl linoleate. The more complex
structure of the starting material has no influence on the reaction
mechanism. The inhibitor that contains conjugated diene
moieties is also expected to be formed. Indeed, the isomerization
of dioleoylphosphatidylcholine in t-BuOH reached the equilib-
rium mixture cis/trans ) 20/80 in ∼100 min, whereas the ratio
cis/trans ) 90/10 is obtained from Figure 6a under the same
conditions.
-
4
methyl linoleate with 1.34 × 10 M oxygen (O); methyl linoleate
with 7 mM all-trans-retinol acetate (]). (b) Time profiles of 9cis,-
12trans-18:2 (3) and 9trans,12cis-18:2 (4) formation without oxygen.
evaporated to a thin film in a test tube under an argon stream
and then kept under vacuum for 30 min. A degassed phosphate-
saline buffer was added, and multilamellar vesicles were formed
by vortex stirring for 7 min under an argon atmosphere. LUVET
were prepared by membrane extrusion with LiposoFast. The
required amounts of HOCH2CH2SH (7 mM) and 2-propanol
Generation of Radicals in Vesicles. In the heterogeneous
system (vesicles), thiols can either be incorporated into the
bilayer or be dissolved in the aqueous phase. Amphiphilic
HOCH2CH2SH and glutathione (GSH) were used without any
concern about the partition of thiol between hydrophobic and
(
0.23 M) were added to LUVET suspensions prior to irradia-
52
tion. The reaction progress was monitored as already described.
The rates of disappearance of oleate, linoleate, and linolenate
moieties are similar to those observed in the isomerization
experiments in t-BuOH. Figure 8a (solid symbols) shows the
behavior of linoleate residues. The yield from the initial fatty
acid composition to the final mixture (after 5 h of irradiation)
was quantitative (by GC analysis whereas TLC showed the
absence of byproducts, such as dimers and adducts). The plot
of G disappearance versus dose for linoleic acid residue and
50
hydrophilic regions. Radiolysis of neutral water leads to the
-
•
•
species eaq , HO , and H as shown in eq 6, where the values
in parentheses represent the yields expressed in terms of G
-1 34a
values (µmol J ). The presence of N2O efficiently transforms
-1
the extrapolation to zero dose gives G = 6 µmol J . This value
-
•
9
-1 -1
eaq into the HO radical (eq 7, k7 ) 9.1 × 10 M s ).
is similar to the one measured in t-BuOH, indicating that the
isomerization process in the heterogeneous system is as efficient
as in homogeneous solution. In the analogous experiments of
dioleoylphosphatidylcholine, a decreased efficiency of the cis-
•
•
Hydrogen abstraction from i-PrOH by HO and H produces
•
9
-1 -1
(
1
CH3)2 COH (eqs 8 and 9, k8 ) 1.9 × 10 M s , k9 ) 7.4 ×
7
-1 -1
•
0 M s ). The (CH3)2 COH in turn reacts with the thiol to
8
give the corresponding thiyl radical (eq 10, k10 ) 5.1 × 10
•
trans isomerization by the HOCH2CH2S radical was observed
-1
-1
8
-1 -1
M
s
for HOCH2CH2SH and k10 ) 1.8 × 10 M
s
for
in going from the homogeneous solution to the LUVET
3
5b
GSH).
24
experiment. Therefore, we suggest that the formation of
conjugated dienes (i.e., the inhibitor) during the isomerization
process of PC in LUVET is less, compared to the analogous
reaction in t-BuOH. However, we observed some substantial
differences between the homogeneous t-BuOH solution and
LUVET experiments: (i) the two mono-trans isomers of linoleic
acid residues are formed in different amounts, the 9trans,12cis-
18:2 being more abundant (Figure 8b), and (ii) the di-trans
isomer (9trans,12trans-18:2) is formed in higher amounts since
the initial phase (Figure 8a, solid squares).
Η Ã
2
eaq- + N O f N + HO + HO
•
-
(7)
(8)
2
2
•
•
HO + (CH ) CHOH f (CH ) COH + H O
3
2
3 2
2
•
•
H + (CH ) CHOH f (CH ) COH + H
(9)
3
2
3 2
2
•
•
(
CH ) COH + RSH f (CH ) CHOH + RS
(10)
3
2
3 2
When the reaction in LUVET was repeated in the presence
of 10% oxygen, no substantial differences were observed.5
3,55
Isomerization of PC in LUVET. As far as the model
membranes are concerned, large unilamellar vesicles (LUVET)
made by the extrusion technique51 were tested by γ-irradiation
using HOCH2CH2SH or GSH as thiols. A PC chloroform
solution (3 mL; 0.15 mmol of fatty acid contents) was
•
•
(
52) 2-Propanol was used in order to avoid reactions of OH and H
species with moieties other than SH in the thiols. The rate constants for the
•
•
reactions of OH and H species with the employed thiols are known to be
close to 1010 and 109 M-1 s-1, respectively.34b To avoid changes in the
lipid aggregates, we could not use 2-propanol as much as was necessary to
•
•
capture all the OH and H. Therefore, on the basis of the available rate
•
•
34b
(
50) Newton, G. L.; Aguilera, J. A.; Kim, T.; Ward, J. F.; Fahey, R. C.
Radiat. Res. 1996, 146, 206-215 and references therein.
51) Liposomes a practical approach; New, R. R. C., Ed.; IRL Press:
Oxford, U.K., 1990.
constants of the OH and H species with 2-propanol and the two thiols,
the 2-propanol concentration was chosen (0.23 and 0.47 M for 7 mM
•
(
HOCH2CH2SH and GSH, respectively) so that ∼90% of the OH radicals
•
and ∼70% of the H atoms reacted with 2-propanol (eqs 8 and 9).

4466 J. Am. Chem. Soc., Vol. 123, No. 19, 2001
Ferreri et al.
The initially formed alkyl radicals from 2-propanol (eqs 8
and 9) in the aqueous compartment react with thiol to form the
corresponding thiyl radical that has to migrate into the lipid
compartment prior to isomerization. For the amphiphilic thiol,
there are no barriers of migration between the aqueous and lipid
compartments. Therefore, the thiyl radical entering the hydro-
phobic region of the bilayer first reaches and isomerizes the
9
,10-double bond. This “positional effect” is the simplest
explanation for the different percentage of the two mono-trans
isomers, the 9trans,12cis-18:2 being the more abundant (see
Figure 8b and 9b). Due to the packing of fatty acid residues
and, consequently, to a highly defined lateral diffusion,56 thiyl
radicals, after the isomerization of the 9,10-double bond, have
a chance to isomerize the second double bond in the same chain,
before migrating laterally.
Figure 9. Irradiation (24.7 Gy min-1) of PC from soybean lecithin in
LUVET (0.15 M of fatty acid contents) with 7 mM GSH at 22 °C. (a)
Unsaturated fatty acid residues vs dose: methyl oleate (O), methyl
elaidate (0), methyl linoleate (b), mono-trans isomers (2), or methyl
linolelaidate (9). (b) Time profiles of 9cis,12trans-18:2 (3) and 9trans,-
As a consequence of the product studies described above and
in view of the well-assessed capability of retinoid and carotenoid
57
molecules to react with thiyl radicals, we have tested the
inhibition of the isomerization process by irradiating LUVET
where 7 mM all-trans-retinol acetate was incorporated into the
lipid bilayer. Figure 8a (open diamonds) shows the dose profile
of the disappearance of linoleate moieties. The plot of G
disappearance of linoleate versus dose gives a G value of ∼1
1
2cis-18:2 (4) formation.
Figure 8a (open circles) shows the dose profile of the dis-
appearance of linoleate moieties. In comparison with the
experiment without oxygen, the isomerization is perhaps slightly
slower. Indeed, a plot of G disappearance of linoleate versus
-1
µmol J by extrapolation of the curve to zero dose that indicates
the absence of a catalytic reaction. Moreover, after 7 h of
irradiation, the overall yield was found to be quantitative. In
comparison with the experiment without all-trans-retinol acetate,
the reaction is substantially retarded. Indeed, the polyene chain,
intercalating into the bilayer, is able to compete with the lipid
double bonds for the thiyl radical addition.
-
1
dose gives G = 5.6 µmol J (by extrapolation of the curve to
-
1
zero dose), which is comparable to G = 6 µmol J obtained
without oxygen. The yield of the reaction slightly dropped (i.e.,
from ∼100 to ∼94%), and TLC showed traces of polar
secondary products such as adducts. However, also in these
experiments, the two mono-trans isomers are present in a
different ratio, the 9trans,12cis-18:2 being the more abundant
Conclusions
(similar to Figure 8b), and the rate of di-trans isomer formation
We have shown that phospholipids containing trans-fatty acid
residues are the major products of the thiyl radical attack on
phosphatidylcholine from soybean lecithin, both in homoge-
neous solution and in lipid vesicles. The presence of oxygen in
concentrations higher than that of typical well-oxygenated
tissues does not influence the reaction outcome, and PUFA
is again faster. Therefore, in LUVET the efficiency of the cis-
trans isomerization without or with 10% oxygen can be
considered the same.
To model the biological environment, the HOCH2CH2SH was
5
2
replaced by GSH. It is gratifying to see that the cis-trans
isomerization is still effective (Figure 9a). For example, the plot
of G disappearance versus dose for linoleic acid residue and
22
peroxidation is found to be unimportant. Our observations are
in good agreement with the known role of “protection” played
-
1
the extrapolation to zero dose gives a G value of 4.5 µmol J .
Comparison of the two experiments with different thiols suggests
that the isomerization rate follows the lipophilicity order of the
22,58
by thiols in autoxidation processes.
The present findings also
test the hypothesis of an endogenous radical-based isomerization
process occurring in the biological environment. This event does
not cause lipid degradation, but a permanent modification, and
deserves attention at least for two aspects: (i) the influence of
trans-fatty acid residues on the structure and properties of human
cell membranes and (ii) the perturbation of cell adaptation
systems, which are based on the number of cis unsaturation
present in the bilayer of bacteria and plants. Lipophilic
compounds containing a polyconjugated dienic moiety inhibit
the cis-trans isomerization process. In this respect, retinoids
and carotenoids that are well known to participate in diverse
5
0
two compounds (i.e., HOCH2CH2SH > GSH). Also in the
experiments with GSH, we observed a similar behavior regard-
ing the formation of mono-trans and di-trans isomers (Figure
9b). Therefore, this phenomenon is not associated with the nature
of the thiol but with the supramolecular organization of
unilamellar vesicles.
-
3
(
53) The solubility of molecular oxygen in H2O is 1.34 × 10 M at 22
54
°
C and 1 atm partial pressure. Therefore, 10% of the oxygen-saturated
solution is equal to 1.34 × 10 M which is ∼3 times higher than a typical
-
4
well-oxygenated tissues, i.e., [O2] = 0.04 mM.
(
5
9a
processes, such as vision, growth, development, and anti-
54) Battino, R.; Rettich, T. R.; Tominaga, T. J. Phys. Chem. Ref. Data
5
9b
1
983, 12, 163-178.
autoxidation, could also be protective agents for the double
bond geometry.
(
55) Thiyl radicals have been reported to add reversibly to oxygen and
•
•
-1
thiolate. For example GS + O2 h GSOO , K ) 3200 M (rate constants
9
-1 -1
5
for the forward and reverse reactions are 2.0 × 10 M
s
and 6.2 × 10
(56) Zachowski, A. Biochem. J. 1993, 294, 1-14.
(57) Everett, S. E.; Dennis, M. F.; Patel, K. B.; Maddix, S.; Kundu, S.
C.; Willson R. L. J. Biol. Chem. 1996, 271, 3988-3994. D’Aquino, M.;
Dunster, C.; Willson, R. L. Biochem. Biophys. Res. Commun. 1989, 161,
1199-1203.
(58) Barclay, L. R. C.; Dakin, K. A.; Khor, J. A. Y. Res. Chem. Intermed.
1995, 21, 467-488.
(59) (a) Handbook of Vitamins: nutritional, biochemical, and clinical
aspects; Machlin, L. J., Ed.; Marcel Dekker: New York, 1984; pp 1-43.
(b) For example, see: Burton, G. W.; Ingold, K. U. Science 1984, 224,
569-573. Samokyszin, V. M.; Marnett, L. J. Free Radical Biol. Med. 1990,
8, 491-496.
-
1
•
-
•-
-1
s
, respectively) and GS + GS h GSSG , K ) 3500 M (rate constants
8 -1 -1 5
for the forward and reverse reactions are 6.2 × 10 M
s
and 1.8 × 10
-
1
s
, respectively). The disulfide radical-anion is known to react with oxygen
8
-1 -1
to give superoxide (k ) 5.1 × 10 M
s ), which might be expected to
oxidize the GSH slowly (k ≈10 M s-1) to regenerate GS radical. Under
3
-1
•
our experimental conditions, these reaction are considered to be unimportant
(
For recent reviews, see: Wardman, P. In S-Centered Radicals; Alfassi,
Z. B., Ed.; Wiley: Chichester, U.K., 1999; pp 289-309. Wardman, P.;
von Sonntag, C. Methods Enzymol. 1995, 251, 31-45). However, work is
in progress to evaluate the efficiency of cis-trans isomerization with
•
-
•
selectively generated species such as O2 and NO2.

Cis-Trans Isomerization of PUFA by Thiyl Radicals
J. Am. Chem. Soc., Vol. 123, No. 19, 2001 4467
Work is in progress in order to evaluate the importance of
cis-trans isomerization in living systems as well as of endog-
enous factors involved in the protection of geometrical lipid
integrity from free-radical attacks.
(100), 261 (40), 243 (50). Further elution gave a third fraction which
was attributed to a mixture of thiol-fatty acid adducts (11 mg, 2%yield;
1
R
(
f
) 0.16): H NMR δ 0.86 (m, 3H), 1.20-1.42 (m, 16H), 1.48-1.61
m, 4H), 1.98 (m, 2H, recognizable as a major q with J ) 6.7 Hz, allyl
CH
CH
2
), 2.12 (m, 1H, allyl CH), 2.23 (m, 1H, CH allyl), 2.27 (m, 3 H,
alpha to carbonyl + OH), 2.59 (m, 1H, CHS), 2.71 (m, 2H, CH S),
O), 5.28-5.48 (m, 2H); C NMR
), 22.2, 22.3, 25.0, 26.5-27.2, 29.1-29.6, 31.4, 31.8, 32.6,
3.8, 34.0, 34.1, 34.4, 34.5, 34.8, 34.9, 38.3 (CH ), 45.1, 45.8 (CH),
1.4 (CH ), 60.6, 60.7 (CH ), 126.1, 126.3, 126.5, 128.9, 129.1, 130.5,
2
2
Experimental Section
13
3.64 (s, 3H, OMe), 3.67 (m, 2H, CH
2
δ 14.2, (CH
3
2 2
Materials. AIBN, AMVN, AAPH, PhSH, HOCH CH SH, GSH,
3
5
1
2
methyl oleate, methyl linoleate, linoleic acid methyl ester cis and trans
isomers, linolelaidic acid methyl ester, linolenic acid methyl ester,
linolenic acid methyl ester isomer mix, conjugated octadecadienoic acid
methyl esters, and methyl palmitate were commercially available from
Aldrich, Fluka, or Sigma and used without further purification.
Lyophilized L-R-phosphatidylcholine from soybean (99%) purchased
from Sigma was dissolved in chloroform (20 mg/mL) and stored in
3
2
30.9, 131.2, 131.9, 132.1, 133.1, 133.3 (CH), 174.0 (CdO); MS m/z
+
(
(
rel abundance): 371 (M +1, 3), 355 (9), 327 (100), 295 (22), 263
9), 243 (46).
Competitive Isomerization of Methyl Oleate and Methyl Li-
noleate. In a Wheaton reactor equipped with a Mininert valve and a
magnetic stirrer, methyl oleate (44.4 mg; 0.15 mmol), methyl linoleate
1-mL sealed ampules at -18 °C. tert-Butyl alcohol, chloroform, and
2-propanol were purchased from Merck (HPLC grade) and used without
(44 mg; 0.15 mmol), and methyl palmitate (27 mg; 0.1 mmol) as the
internal standard were dissolved in 1 mL of t-BuOH. The solution was
degassed with argon for 15 min, added with AMVN (7.5 mg; 0.03
further purification. Phosphate saline buffer (PBS) was prepared (Na
2
-
HPO 10 mM, NaCl 0.14 M) at pH 7.2.
4
mmol) and HOCH
2 2
CH SH (5.86 mg; 0.075 mmol), and then heated at
General Methods. GC analyses for the determination of the isomeric
ratio of the unsaturated fatty acids were performed by using a Varian
CP-3800 equipped with a flame ionization detector. As a stationary
phase, an Rtx-2330 column (60 m × 0.25 mm of 10% cyanopropyl-
phenyl and 90% biscyanopropylpolysiloxane) was used with helium
as carrier gas. The heating was carried out at a temperature of 155 °C
for 55 min followed by an increase of 5 °C/min up to 195 °C. The
methyl esters were identified by comparison with the retention times
of authentic samples. In few cases, GC analyses were performed on an
HP 5890 Series II using a 30 m × 0.25 mm cross-linked 5%
phenylsilicone capillary column (HP 5).
5
4 °C. At different times, an aliquot of the reaction mixture was
withdrawn for the GC analysis.
Isomerization of Methyl Linolenate in tert-Butyl Alcohol. Condi-
tions similar to the above-mentioned isomerization of methyl linoleate
were used. After 7 h of irradiation, a 92% yield of the eight isomers
was found. A TLC examination of the reaction mixture (eluent, 7/3
n-hexane/diethyl ether) evidenced the formation of two secondary
products, which, in analogy with the byproducts isolated from the
linoleate reaction, were attributed to dimers (R
fatty acid adducts (R ) 0.11).
f
) 0.57) and to thiol-
f
The reaction mixture was evaporated and flash chromatographed.
Elution with n-hexane gave a first fraction which contained methyl
palmitate and a mixture of the eight 18:3 isomers in a 92% yield. Further
elution with 9/1 n-hexane/diethyl ether gave a second fraction which,
from the spectral characteristics, was attributed to a mixture of dimeric
conjugated fatty acids (4.0 mg; 4% yield), whereas elution with 1/1
n-hexane/diethyl ether gave a third fraction (2 mg; 3.6% yield) which,
from the spectral characteristics, was attributed to a mixture of thiol
addition products.
Isomerization of L-r-Phosphatidylcholine from Soybean Lecithin
in tert-Butyl Alcohol. A PC/chloroform solution (3 mL; 0.15 mmol
of fatty acid content) was evaporated in a test tube under an argon
stream and then under vacuum for 30 min. t-BuOH (1 mL) was added,
and the solution was transferred to a vial equipped with an open top
Continuous radiolyses were performed at room temperature (22 (
60
2
°C) on 1-mL samples using a Co Gammacell at different dose rates.
The exact absorbed radiation dose was determined with the Fricke
3
+
-1 60
chemical dosimeter, by taking G(Fe ) ) 1.61 µmol J
.
The flow
monitoring of a 1:1 N O/air mixture prior to γ-irradiation was controlled
2
3
-1
by a flowmeter to be at 68 cm min . Occasionally the gas flowing
was continued during the irradiation by means of a cannula.
Flash chromatography was performed on Merck silica gel 60 (400-
1
13
2
30 mesh) using a nitrogen stream. H, C NMR spectra were recorded
on a Varian VXR 400-MHz instrument using CDCl as the solvent
3
and the reference peak. EI MS spectra were recorded on a Finnigan
MAT GCQ instrument equipped with a direct insertion probe DIP.
Isomerization of Methyl Linoleate in tert-Butyl Alcohol. A 0.15
M solution of methyl linoleate (43 mg) in t-BuOH (0.945 mL) was
placed under argon in a 2.5-mL vial equipped with an open top screw
cap and a Teflon-faced septum and added with methyl palmitate (27
mg; 0.1 mmol) as the internal standard. The solution was degassed
with argon for 15 min, added with 0.03 M AMVN, 0.075 M PhSH, or
screw cap and a Teflon-faced septum where it was saturated with N
or a 1:1 N O/air mixture prior to γ-irradiation. HOCH CH SH (0.075
mmol) was then added, and the solution was irradiated. Aliquots of
00 µL were withdrawn and processed at different times by partitioning
2
O
2
2
2
1
between n-hexane (or 2/1chloroform/methanol in the case of vesicles)
and brine, extraction and collection of the organic phases dried over
anhydrous sodium sulfate, and evaporation of the solvent under vacuum
at room temperature. The residue containing the phospholipids was
treated with 0.5 M KOH/MeOH, for 10 min at room temperature and
then poured into the brine and extracted with n-hexane. The organic
layer containing the corresponding fatty acid methyl esters was
examined by GC analysis in comparison with the retention times of
authentic samples.
Isomerization of PC in LUVET. A chloroform solution of PC (3
mL; 0.15 mmol of fatty acid content) was evaporated to a thin film in
a test tube under an argon stream and under vacuum for 30 min.
Degassed PBS (1 mL) was added, and multilamellar vesicles were
formed by vortex stirring for 7 min under an argon atmosphere. To
obtain LUVET, the lipid emulsion was transferred into a LiposoFast
HOCH
Alternatively, the solution was saturated with N
mixture, followed by the addition of HOCH CH SH prior to γ-irradia-
tion. Aliquots of the solution were withdrawn at different times and
examined by GC analysis.
2
CH
2
SH and a magnetic stirrer, and then heated at 54 °C.
2
O or a 1:1 N O/air
2
2
2
The isomerization of linolelaidic acid methyl ester and linoleic acid
methyl ester isomer mix was performed under similar conditions.
Product Studies in Methyl Linoleate Isomerization with HOCH
2
-
CH SH. The isolation of the products was effected using a 10-mL scale
2
of the above-reported experiment after 7 h of irradiation. The reaction
mixture was evaporated under vacuum and flash chromatographed.
Using n-hexane as the eluent, a first fraction was obtained corresponding
to a 93% yield of the four geometrical isomers. Elution with 8/2
n-hexane/diethyl ether gave a second fraction, which was attributed to
a mixture of dimeric conjugated fatty acids (18 mg, 4% yield; R )
f
(produced by Avestin, Inc.) and extruded 19 times back and forth
1
0
4
5
2
.47): H NMR δ 0.88 (m, 6H), 1.05-1.48 (m, 32H), 1.56-1.63 (m,
through two polycarbonate membranes with a pore diameter of 100
nm.61 The suspension was then transferred to a vial equipped with an
open top screw cap and a Teflon-faced septum where it was saturated
H), 1.92-2.05 (m, 6H, allylic Hs), 2.26-2.32 (m, 4H), 3.66 (s, 6H),
1
3
3
.14-6.24 (m, 8H); C NMR δ 13.9, 14.2 14.3 (CH ), 22.6, 22.7,
5.0, 27.1-27.9, 29.1-29.7, 30.6, 31.6, 31.8, 32.0, 32.1, 32.7, 34.2
with N
2
2 2 2
O or a 1:1 N O/air mixture prior to γ-irradiation. HOCH CH -
(CH
2
), 40.6, 47.6, 47.9, 48.1, 48.3 (CH), 51.5 (CH
3
), 128.6-134.3 (CH),
+
1
74.1 (CdO); MS m/z (rel abundance): 586 (M , 3), 555 (10), 293
(61) The average diameter of the unilamellar vesicles was found to be
90 nm; see: Fiorentini, D.; Cipollone, M.; Pugnaloni, A.; Biagini, G.;
Landi, L. Free Radical Res. 1994, 21, 329-339.
∼
(60) Reference 32; p 100.

4468 J. Am. Chem. Soc., Vol. 123, No. 19, 2001
Ferreri et al.
SH or GSH (0.075 mmol) and i-PrOH (0.23 or 0.47 M, respectively)
were consecutively added and the suspension was irradiated. Aliquots
of 100 µL were withdrawn and processed at different times as
previously described.
Isomerization of PC in LUVETs in the Presence of Vitamin A.
all-trans-Retinol acetate (23 mg; 0.07 mmol) was dissolved in
chloroform (1 mL), and 100 µL of this stock solution (corresponding
to 7 µmol of vitamin A acetate) was evaporated in a test tube under an
argon stream. A chloroform solution of PC (3 mL; 0.15 mmol of fatty
acid content) was then added and evaporated to a thin film under an
argon stream and under vacuum for 30 min. LUVETs were prepared
and irradiated as previously described.
Acknowledgment. We thank Professor Christian Sch o¨ neich
for helpful discussions. Financial support from MURST and
the University of Bologna (research project “Free radicals and
radicals ions in chemical and biological processes” to C.F. and
L.L.) is gratefully acknowledged.
JA0040969