Reaction of retinol with peroxynitrite

Article abstract of DOI:10.1271/bbb.70349

The reactivity of retinol with peroxynitrite, which is a strong oxidant and has been reported to induce several biological damages, was investigated. 13-cis-14-nitroretinol (1), 13-trans-14-nitroretinol (2), 13-apo-β- carotenone (3), retinal (4), 11,14-epoxyretinol (5), and 11,15-epoxyretinol (6) were identified as reaction products of retinol with peroxynitrite. From these results, it was observed that retinol can undergo a nitration reaction with peroxynitrite. Furthermore, the formation mechanisms of 1, 2, and 3 from retinol with peroxynitrite are discussed.

Full text of DOI:10.1271/bbb.70349

Biosci. Biotechnol. Biochem., 71 (10), 2596–2599, 2007
Note
Reaction of Retinol with Peroxynitrite
2;
1
1
Rie SUZUKI,1; Aditya KULKARNI,
Yuya YOMODA, Hirokazu KAWAGISHI,
*
*
y
Yukimasa TERADA,3 Takashi MAOKA,4 and Hideo ETOH
1;
1Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
2United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
3Center of Computers and Technology, Meijo University, Shiogamaguchi, Tempaku, Nagoya 468-8502, Japan
4Research Institute for Production Development, 15 Shimogamo-Morimoto-cho, Sakyou-ku, Kyoto 606-0805, Japan
Received June 4, 2007; Accepted July 18, 2007; Online Publication, October 7, 2007
[doi:10.1271/bbb.70349]
The reactivity of retinol with peroxynitrite, which is a
strong oxidant and has been reported to induce several
biological damages, was investigated. 13-cis-14-nitro-
retinol (1), 13-trans-14-nitroretinol (2), 13-apo-ꢀ-caro-
tenone (3), retinal (4), 11,14-epoxyretinol (5), and 11,15-
epoxyretinol (6) were identified as reaction products of
retinol with peroxynitrite. From these results, it was
observed that retinol can undergo a nitration reaction
with peroxynitrite. Furthermore, the formation mech-
anisms of 1, 2, and 3 from retinol with peroxynitrite are
discussed.
peroxynitrite. Here we discuss the formation mecha-
nisms of the above reaction products.
All-trans-retinol (150 mg) was dissolved in 100 ml of
THF (final concentration, 4.8 mM). To this, 200 ml of
TFA (final concentration, 0.2%) was added, followed by
10 ml of peroxynitrite, which was prepared according to
a method described in the literature9) (final concentration
4.5 mM), and allowed to react for 2 min. To the above
mixture, 200 ml of chloroform and 200 ml of H2O were
added, so as to separate the reaction products into
organic and aqueous phases. The entire procedure was
performed two times.
Key words: retinol; peroxynitrite; antioxidative com-
pounds
The organic layer was then dehydrated using anhy-
drous Na2SO4 and concentrated. This organic concen-
trate was subjected to HPLC analysis with a semi-
prepatative C30 column (Develosil C30-UG-5 ꢁ20 ꢀ
250 mm, Nomura Chemical Co., Ltd., Seto, Japan) with
65% MeCN as the mobile phase. For more specific
separation, an analytical C30 column was used (Devel-
osil C30-UG-5 ꢁ4:6 ꢀ 250 mm, Nomura Chemical Co.,
Ltd., Seto, Japan) with 65% MeCN and seven fractions
were obtained, as shown in Fig. 1.
Compounds 1, 2, 3, 4, 5, and 6 were identified as
retinal (3.8%) (4), 13-apo-ꢀ-carotenone (3.6%) (3),10)
13-cis-14-nitroretinol (3.1%) (1), 13-trans-14-nitroreti-
nol (2.1%) (2), 11,15-epoxyretinol (1.5%) (6),11,12) and
11,14-epoxyretinol (1.3%) (5), by MS, UV–VIS, 1H
and 13C NMR data and by comparison with authentic
samples (Fig. 2). Among these, 13-cis-14-nitroretinol
and 13-trans-14-nitroretinol13) were identified for the
first time as major products of this reaction. In order to
determine the stereochemistry of 1 and 2, NOESY
studies were performed. NOE interaction was observed
between 20-CH3 and 14-H of 1, indicating cis isomer-
ism, but no NOE interaction was observed between 20-
CH3 and 14-H in case of 2, confirming it to be a trans
isomer of nitroretinol. Compounds 5 and 6 were
identified as mixtures, since they showed the same
Retinol, a circulating form of vitamin A, is a fat-
soluble molecule that plays an important role in the
visual cycle and is essential for the normal growth of
bones, reproduction, and embryonic development and in
the differentiation of epithelial tissues. It is generated
from either ꢀ-carotene or retinyl esters and is stored as
retinyl esters in the liver. Retinols are sensitive to
oxidation.1–4) The extended polyene structure of retinol
readily reacts with the peroxyl radical and is thought to
inhibit lipid peroxydation by trapping lipid-peroxy
radicals.5) Peroxynitrite is a strong oxidant and has the
potential to cause several forms of biological damage.
Peroxynitrite and its decomposition products induce
DNA strand scission, protein modification by nitration
and hydroxylation, and membrane lipid peroxydation in
LDL,6,7) but the peroxynitrite reaction mechanism of
retinol, which probably figures in the human body, is not
yet understood. In our recent efforts, we isolated for the
first time nitro carotenoids from the reaction of
astaxanthin and ꢀ-carotene with peroxynitrite.8) In our
continuous efforts in this field, we investigated the
reactivity of retinol with peroxynitrite and attempted to
identify its reaction products after scavenging with
*
The two authors contributed equally to this work.
To whom correspondence should be addressed. Tel/Fax: +81-54-238-4884; E-mail: acheto@agr.shizuoka.ac.jp
y

Reaction of Retinol with Peroxynitrite
2597
Fig. 1. HPLC Chromatogram of Peroxynitrite Reaction of Retinol.
HPLC conditions: C30, Develosil C30-UG-5 ꢁ4:6 ꢀ 250 mm, 65% MeCN, flow rate; 1 ml/min, column temperature, 40 ꢁC, detection range,
200–600 nm.
20
15
13
OH
14
Retinol
20
20
13
13
NO2
H
14
12
14
12
H
NO2
13-trans-14-nitroretinol (2)
13-cis-14-nitroretinol (1)
20
H
11
13
O
H
O
10
12
retinal (4)
13-apo-β -carotenone (3)
15
15
14
OH
O
14
O
11
OH
11
13
20
10
13
12
20
10
12
11,14-epoxyretinol (5)
11,15-epoxyretinol (6)
O
1
7
8
(H3C)3C
C(CH3)3
2
6
5
3
4
O
9
O
4'
1'
2'
3'
BHT-THF adduct
Fig. 2. Structures of Peroxynitrite Reaction Products of Retinol.

2598
R. SUZUKI et al.
20
13
15
OH
14
Nitronium radical pathway
15
Dioxetane pathway
Retinol
CHO
20
13
Retinal
14
15
OH
14
15
O
O
COOH
Retinoic acid
14
NO2
(ONOOH*)
13, 14-dioxetane retinol
20
O
11
13
15
13
C
O
O
14
10
12
NO2
20
3
13
14
NO2
1
20
13
NO2
14
2
Fig. 3. Reaction Mechanism.
retention time in HPLC chromatograms (Fig. 1). The
peak at a retention time of 34 min corresponded to the
reaction product between THF and BHT. THF contained
a trace amount of BHT as a stabilizer, which might
undergo a radical reaction in presence of peroxynitrite to
form the adduct. The taller peak at a retention time of
55 min was of unreacted retinol.
13-cis-14-nitroretinol (1) UV ꢂmax (CH3CN): 427 nm;
high resolution (HR) FAB-MS calculated for C19H27-
O2N + Hþ = 301.2042, found = 301.2038; yield 3.1%
1H-NMR ꢃ (CDCl3): 0.97 (6H, s, 16, 17-CH3), 1.41 (2H,
m, 2-CH2), 1.56 (2H, m, 3-CH2), 1.66 (3H, s, 18-CH3),
1.97 (3H, s, 19-CH3), 1.98 (2H, m, 4-CH2), 2.03 (3H, s,
20-CH3), 6.14 (1H, d, J ¼ 16 Hz, 8-CH), 6.24 (1H, d,
J ¼ 12 Hz, 10-CH), 6.33 (1H, d, J ¼ 16 Hz, 7-CH), 6.85
(1H, s, 14-CH), 7.17 (1H, dd, J ¼ 12 Hz, 15 Hz, 11-CH),
7.69 (1H, d, J ¼ 15 Hz, 12-CH).
7-CH), 7.13 (1H, s, 14-CH), 7.23 (1H, dd, J ¼ 11 Hz,
15 Hz, 11-CH), 6.22 (1H, d, J ¼ 15 Hz, 12-CH).
1
BHT-THF adduct H-NMR ꢃ (CDCl3): 1.21 (6H, s,
7,8-(CH3)6), 1.33 (3H, s, 9-CH3), 6.46 (1H, d, 3 or 5-
CH), 6.61 (1H, d, 5 or 3-CH), 1.60 (1H, m, 20-CH), 1.71
(1H, m, 30-CH), 3.84 (1H, q, J ¼ 15 Hz, 8 Hz, 40-CH),
3.90 (1H, sp, J ¼ 4 Hz, 8 Hz, 40-CH), 5.35 (1H, d, J ¼
8 Hz, 10-CH). 13C-NMR ꢃ (CDCl3): 23.9 (9-C), 24.1 (30-
C), 29.1 (20-C), 29.4 (7 and 8-(C)3), 34.7 (7 and 8-C),
67.5 (40-C), 78.0 (4-C), 107.2 (10-C), 129.1 (5-C), 140.5,
141.2 (3 and 5-C), 147.2, 147.6 (2 and 6-C), 186.5 (1-C).
From the results of the present investigation, we
summarized the possible reaction mechanisms of per-
oxynitrite with retinol (Fig. 3) and proposed formation
pathways for 13-cis (1) and 13-trans-nitroretinol (2)
(nitronium radical pathway), as follows: Retinoic acid, a
possible intermediate, was formed from retinol via
retinal (4) by an oxidation reaction. Then retinoic acid
was reacted with the (ꢂNO2) or (ONOOHꢃ) radical at the
C-14 position to yield 13-cis- and 13-trans-14-nitro-
retinol as major products. We did not detect retinoic acid
as an intermediate product of this reaction, but we car-
ried out a reaction between retinoic acid and peroxyni-
trite separately, which gave us the expected reaction
products 13-cis (1) and 13-trans-14-nitroretinol (2).
13-trans-14-nitroretinol (2) UV ꢂmax (CH3CN): 419
nm; high resolution (HR) FAB-MS calculated for C19-
H27O2N + Hþ = 301.2042, found = 301.2042; yield
2.1%. 1H-NMR ꢃ (CDCl3): 1.04 (6H, s, 16,17 CH3),
1.48 (2H, m, 2-CH2), 1.58 (2H, m, 3-CH2), 1.72 (3H, s,
18-CH3), 2.04 (3H, s, 19-CH3), 2.05 (2H, m, 4-CH2),
2.45 (3H, s, 20-CH3), 6.18 (1H, d, J ¼ 17 Hz, 8-CH),
6.32 (1H, d, J ¼ 11 Hz, 10-CH), 6.38 (1H, d, J ¼ 17 Hz,

Reaction of Retinol with Peroxynitrite
2599
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the trans isomer, and this agreed quantitatively with the
yield of these isomers (3.1%, for 1 and 2.1%, for 2).
For the formation of 13-apo-ꢀ-carotenone (3), we
propose the Dioxetane pathway as follows: Retinols
oxidize to 13, 14-dioxetane of retinol, a possible
intermediate, by the oxygen radical, which might form
during the reaction of peroxynitrite with retinol. Then
oxidative cleavage between C-13 and C-14 of 13, 14-
dioxetane retinol results in the formation of 13-apo-ꢀ-
carotenone (3) as one of the main products.
In conclusion, we have for the first time isolated and
characterized nitroretinols 1 and 2 from the reaction
products of peroxynitrite with retinal, and, we propose
the formation mechanism. These results indicate that
retinol caught the peroxynitrite or nitrogen dioxide
radical (ꢂNO2) in its molecule to form nitroretinols. This
information should be of value in the future for
understanding certain complex in vivo reaction patterns
of retinol and its oxidation products. Efforts are under-
way to elucidate the structure of intermediates and
products by way of understanding the complete mech-
anism of scavenging peroxynitrite.
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Giraud, M., Labia, R., and Potier, P., Syntheses of new
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Acknowledgments
This work was supported by the program Cooperation
of Innovative Technology and Advanced Research in the
Evolutional Area (Metropolitan Regions) of the Ministry
of Education, Culture, Sports, Science and Technology
of Japan.
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Peroxyl-radical reaction of retinyl acetate in solution.
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Epoxidation of retinol and the structure of chromogen
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under mildly acidic conditions: toward a reappraisal of
retinoids as scavengers of reactive nitrogen species.
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14) Ab initio calculations were carried out using the
Gaussian 03 program (Gaussian, Wallingford, CT,
2004). The starting coordinates were generated by
inspection of the molecular model, and then they were
fully optimized using RHF/6-31G (D). The calculations
were carried out using a Fujitsu Primepower HPC2500
super computer at the Information Technology Center of
Nagoya University.
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