Occurrence of cytotoxic 9-oxononanoyl secosterol aldehydes in human low-density lipoprotein

Article abstract of DOI:10.1016/j.freeradbiomed.2013.01.029

The reaction products of three major cholesteryl esters, cholesteryl palmitate (C16:0-CE), cholesteryl oleate (C18:1-CE), and cholesteryl linoleate (C18:2-CE), present in human low-density lipoprotein (LDL) treated with ozone were isolated and characterized. In vitro ozonization of C16:0-CE was found to form the palmitoyl ester of secosterol-A (3β-hydroxy-5-oxo-5,6- secocholestan-6-al) and its aldolization product secosterol-B (3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde). On the other hand, when C18:1-CE and C18:2-CE were oxidized by ozone, the aldehyde 9-oxononanoyl cholesterol (9-ONC) was formed as a primary product, which was then further oxidized to form 9-oxononanoyl secosterol-A (9-ON-secoA) and 9-oxononanoyl secosterol-B (9-ON-secoB). The compounds 9-ON-secoA and -B, but not 9-ONC, were found to exhibit strong cytotoxicity against human leukemia HL-60 cells. An LC-ESI-MS/MS method was developed for the detection of these cholesteryl ester ozonolysis products by derivatizing them with dansyl hydrazine. Using this method, we found for the first time that low concentrations of 9-ON-secoA and -B, but not palmitoyl secosterols, were present in human LDL. These novel oxidized cholesterol esters, 9-ON-secoA and -B, probably play important roles in the pathogenesis of several inflammatory disorders such as cancer, diabetes, atherosclerosis, and neurodegenerative diseases.

Full text of DOI:10.1016/j.freeradbiomed.2013.01.029

Author’s Accepted Manuscript
Occurrence of cytotoxic 9-oxononanoyl secosterol
aldehydes in human low density lipoprotein
Noriyuki Miyoshi, Nozomi Iwasaki, Susumu Tomo-
no, Tatsuya Higashi, Hiroshi Ohshima
www.elsevier.com/locate/freeradbiomed
PII:
S0891-5849(13)00039-7
DOI:
Reference:
http://dx.doi.org/10.1016/j.freeradbiomed.2013.01.029
FRB11448
To appear in:
Free Radical Biology and Medicine
Received date: 11 October 2012
Revised date: 8 January 2013
Accepted date: 29 January 2013
Cite this article as: Noriyuki Miyoshi, Nozomi Iwasaki, Susumu Tomono, Tatsuya
Higashi and Hiroshi Ohshima, Occurrence of cytotoxic 9-oxononanoyl secosterol
aldehydes in human low density lipoprotein, Free Radical Biology and Medicine, http://d
x.doi.org/10.1016/j.freeradbiomed.2013.01.029
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Occurrence of cytotoxic 9-oxononanoyl secosterol aldehydes in human low density
lipoprotein
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Noriyuki Miyoshia, §, *, Nozomi Iwasakia, §, Susumu Tomonoa, Tatsuya Higashib, Hiroshi
Ohshimaa
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aLaboratory of Biochemistry, Graduate School of Integrated Pharmaceutical and Nutritional
Sciences, Graduate Program in Food and Nutritional Sciences, University of Shizuoka,
Shizuoka 422-8526, Japan
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bFaculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba 278-8510,
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Japan
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Running title: Acyl esters of secosterol aldehydes
§ Equally contributed to this work.
*Correspondence to be addressed to: Noriyuki Miyoshi, Ph.D, 52-1 Yada, Suruga-ku,
Shizuoka 422-8526, Japan. Tel.: +81-54-264-5536; Fax: +81-54-264-5530; E-mail:
miyoshin@u-shizuoka-ken.ac.jp
The following abbreviations are used: LDL, low density lipoprotein; secosterol-A,
3ꢀ-hydroxy-5-oxo-5,6-secocholestan-6-al;
secosterol-B,
C16:0-CE, cholesteryl
3ꢀ-hydroxy-5ꢀ-hydroxy-B-norcholestane-6ꢀ-carboxaldehyde;
palmitate; C18:1-CE, cholesteryl oleate; C18:2-CE, cholesteryl linoleate; 9-ONC,
9-oxononanoyl cholesterol; 9-ON-secoA, 9-oxononanoyl secosterol-A; 9-ON-secoB,
9-oxononanoyl secosterol-B; 5-OVA, 5-oxovaleroyl cholesterol; ROS, reactive oxygen
species; DH, dansyl hydrazine; MRM, multiple reaction monitoring
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Abstract
The reaction products of three major cholesteryl esters present in human low
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density lipoprotein (LDL), cholesteryl palmitate (C16:0-CE), cholesteryl oleate (C18:1-CE),
and cholesteryl linoleate (C18:2-CE), treated with ozone were isolated and characterized. In
vitro ozonization of C16:0-CE was found to form the palmitoyl ester of secosterol-A
(3ꢀ-hydroxy-5-oxo-5,6-secocholestan-6-al) and its aldolization product secosterol-B
(3ꢀ-hydroxy-5ꢀ-hydroxy-B-norcholestane-6ꢀ-carboxaldehyde). On the other hand, when
C18:1-CE and C18:2-CE were oxidized by ozone, the aldehyde 9-oxononanoyl cholesterol
(9-ONC) was formed as a primary product, which was then further oxidized to form
9-oxononanoyl secosterol-A (9-ON-secoA) and 9-oxononanoyl secosterol-B (9-ON-secoB).
The compounds 9-ON-secoA and -B, but not 9-ONC, were found to exhibit strong
cytotoxicity against human leukemia HL-60 cells. An LC-ESI-MS/MS method was
developed for the detection of these cholesteryl ester ozonolysis products by derivatizing
them with dansyl hydrazine. Using this method, we found for the first time that low
concentrations of 9-ON-secoA and -B, but not palmitoyl secosterols, were present in human
LDL. These novel oxidized cholesterol esters, 9-ON-secoA and -B, likely play important
roles in the pathogenesis of several inflammatory disorders such as cancer, diabetes,
atherosclerosis and neurodegenerative diseases.
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Key words: LDL, cholesteryl ester, secosterol, LC-ESI-MS/MS, cytotoxicity.
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INTRODUCTION
Low density lipoprotein (LDL) particles contain cholesteryl esters and
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triacylglycerols surrounded by a shell composed of phospholipids, free cholesterol and
apolipoprotein B100. LDL particles are prone to oxidative modifications caused by
enzymatic attack of myeloperoxidase and lipoxygenase, or by reactive oxygen species
(ROS) such as hypochlorous acid (HOCl), phenoxyl radical intermediates or peroxynitrite
generated in the innermost layer of the artery during inflammation and atherosclerosis. The
peroxidation of fatty acid residues in phospholipids, cholesteryl esters, and triacylglycerols
generates reactive aldehydes and truncated lipids. They play important roles in the early
development of atherosclerosis through the recruitment of monocyte-derived macrophages
into the arterial wall, and by promoting the intracellular accumulation of cholesteryl esters
in these cells, resulting in the formation of foam cells [1]. In the necrotic core of an
atherosclerotic lesion, dying foam cells can release their contents, oxidized cholesteryl
esters and other lipid oxidation products, which then accumulate in the extracellular space.
Acyl chain oxidations of cholesteryl esters lead to the formation of corresponding lipid
hydroperoxides, which then undergo carbon-carbon cleavage, yielding aldehydes which are
still esterified to the parental lipid, termed a core-aldehyde [2]. 5-Oxovaleroyl cholesterol
(5-OVA) and 9-oxononanoyl cholesterol (9-ONC) have been reported as two major
core-aldehydes derived from the oxidation of cholesteryl arachidonate and of cholesteryl
linoleate and oleate, respectively [2]. Some of them are thought to result from cellular
damage, whereas others may play important physiological roles in a variety of cellular
functions, including the regulation of cholesterol homeostasis. In addition, cholesterol is
also endogenously oxidized enzymatically or non-enzymatically to form various oxidized
cholesterols [3]. The enzymatic oxidations are mainly performed by the cytochrome P450
superfamily, resulting in the formation of 24S-hydroxy (24S-OH) cholesterol, 27-OH
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cholesterol, and 25-OH cholesterol. The non-enzymatic oxidation products
7-ketocholesetrol, 7ꢀ-OH cholesterol, and 7ꢁ-OH cholesterol are mainly formed by the
direct attack of various ROS on the ring structure. 7ꢁ-OH cholesterol and 25-OH
cholesterol are formed via both enzymatic and non-enzymatic reactions. Secosterol-A
(3ꢀ-hydroxy-5-oxo-5,6-secocholestan-6-al) and its aldolization product secosterol-B
(3ꢀ-hydroxy-5ꢀ-hydroxy-B-norcholestane-6ꢀ-carboxaldehyde) have been recently reported
to be major cholesterol oxidation products formed by cholesterol ozonolysis [4-6]. Elevated
levels of secosterol-A and -B have been detected in human atherosclerotic plaques [7] and
brain tissues of neurodegenerative diseases, such as Alzheimer’s disease and Lewy body
dementia [8, 9]. Secosterols exert strong cytotoxic effects toward various culture cells [7,
10-12] and have the ability to covalently modify proteins, such as the p53, amyloid-ꢀ
peptide and ꢁ-synuclein to accelerate amyloidogenesis and fibrilization [9, 13-15]. We have
reported the mechanism of secosterol-A and -B formation in phorbol myristate acetate
(PMA)-activated HL-60 cell cultures, which was found to be myeloperoxidase-dependent
[16]. Moreover, we revealed that secosterol-A can inhibit activities of endothelial nitric
oxide synthase (NOS) and neuronal NOS, but not inducible NOS [17]. On the basis of these
findings, secosterols have been postulated to play pivotal roles in the pathogenesis of
atherosclerosis and neurodegenerative diseases. Cholesterol can be present in various
tissues as its free form or acyl esters, the latter compounds are present in much higher
concentrations than the free one, the ratio of free cholesterol and its acyl esters is estimated
at 3:7 in the blood [18]. These cholesteryl esters can be oxidized similarly to free
cholesterol, resulting in the formation of various oxidation products. However no studies
have been reported concerning physiological levels and/or bioactivities of these
compounds.
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In this study, we have characterized the ozonolysis products of cholesteryl esters
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and developed a sensitive and specific method to analyze these compounds in biological
specimens. Using this method, we were able to detect 9-oxononanoly secosterol-A and -B
(9-ON-secoA and 9-ON-secoB, respectively) for the first time in healthy human LDL. We
also found that 9-ON-secoA and -B, but not 9-ONC, exert potent cytotoxicity in HL-60
cells, as reported for secosterol-A and -B. Our findings suggest that 9-ON-secoA and -B
may play important roles in the development of several inflammation-related diseases such
as cancer, diabetes, atherosclerosis and neurodegenerative disease.
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MATERIALAND METHODS
Materials
Cholesterol, cholesteryl palmitate (C16:0-CE), cholesteryl oleate (C18:1-CE), and
cholesteryl linoleate (C18:2-CE) were purchased from Sigma-Aldrich, St. Louis, MO.
Dansyl hydrazine (DH) and RPMI 1640 medium were purchased from Invitrogen (Carlsbad,
CA) and Nissui Pharmaceutical Co., Ltd., Tokyo, Japan, respectively. All other chemicals
were obtained from Wako Pure Chemical Industries, Osaka, Japan.
Preparation of secosterol-A and -B
Secosterol-A and -B and 3,4-13C-secosterol-B, as an internal standard for
LC-ESI-MS/MS analyses, were synthesized as reported previously [19]. The purity of the
product was verified by TLC and ¹H-NMR. The stock solutions (10 mM) of secosterols
were prepared in ethanol and stored at -20°C until use.
Preparation of secosterol esters
A solution of cholesteryl palmitate (500 mg, 0.8 mmol) was prepared in a mixture
of chloroform/methanol (9:1) (100 ml). The solution was cooled to dry ice temperature and
ozone (210 μmol/min) was bubbled through the sample for 10 min. The reaction mixture
was evaporated under reduced pressure and stirred with Zn powder (650 mg, 10 mmol) in
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water/acetic acid (1:9) (50 ml) at room temperature for 3 h and diluted with
dichloromethane (100 ml). The mixture was then washed with water (3 × 50 ml), dried over
anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was
purified by silica gel chromatography (EtOAc/n-hexane (1:6)) to give the compound
(5-oxo-5,6-secocholestan-6-al-3ꢀ-palmitate, C16:0-secoA) as a white solid (104.7 mg,
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20%). Data for C16:0-secoA: Rf = 0.4 (EtOAc/n-hexane (1:6)); H NMR (400 MHz,
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CDCl3) ꢂ: 9.61 (s, 1H, CHO-7), 5.38 (s, 1H, H-3), 3.06 (dd, J=14.4, 4.4 Hz, 1H, H-4); 13C
NMR (100 MHz, CDCl3) ꢂ: 216.08 (C-5), 202.64, 172.99, 73.05, 56.12, 54.12, 52.33,
44.04, 43.27, 42.54, 42.09, 39.85, 39.47, 35.97, 35.75, 34.81, 34.48, 34.43, 31.93, 29.69,
29.67, 29.60, 29.45, 29.37, 29.24, 29.07, 28.01, 27.83, 25.15, 24.93, 23.75, 23.08, 22.80,
22.70, 22.55, 18.56, 17.64, 14.13, 11.52; DART-TOF/MS (positive) calcd for C43H77O4 m/z
[M+H]+ 657.5822, found 657.5807. Aldolization of C16:0-secoA. A solution of
C16:0-secoA (53.8 mg, 0.08 mmol) in acetonitrile/water (20:1) (100 ml) was added to
L-proline (24 mg, 0.21 mmol) and stirred for 2 h in room temperature. The mixture was
evaporated under reduced pressure and dissolved in EtOAc (50 ml) and washed with water
(3 × 50 ml). The combined organic layers were dried over anhydrous sodium sulfate, and
concentrated under reduced pressure. The residue was purified by silica gel
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chromatography
(EtOAc/n-hexane
(1:6))
to
give
the
compound
(5ꢀ-hydroxyl-B-norcholestane-6ꢀ-carboxaldehyde-3ꢀ-palmitate, C16:0-secoB) as a white
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solid (34.3 mg, 65%). Data for C16:0-secoB: Rf = 0.4 (EtOAc/n-hexane (1:6)); H NMR
(400 MHz, CDCl3) ꢂ: 13C NMR (400 MHz, CDCl3) ꢂ: 9.68 (d, J=3.0 Hz, 1H, CHO-7), 5.13
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(m, 1H, H-3), 2.51 (s, 1H, OH-5); C NMR (100 MHz, CDCl3) ꢂ: 203.83, 172.47, 83.66
(C-5), 70.15, 64.51, 56.04, 55.67, 51.38, 45.62, 44.67, 42.32, 39.65, 39.48, 39.25, 36.19,
35.62, 34.66, 31.93, 29.67, 29.64, 29.58, 29.45, 29.37, 29.23, 29.10, 28.35, 28.01, 27.96,
25.01, 24.66, 24.44, 23.82, 22.80, 22.70, 22.55, 21.57, 18.75, 18.34, 14.13, 12.50;
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DART-TOF/MS (positive) calcd for C43H77O4 m/z [M+H]+ 657.5822, found 657.5869.
Using the above procedures, cholesteryl oleate (500 mg, 0.77 mmol) or cholesteryl
linoleate (600 mg, 0.92 mmol) was oxidized with ozone to synthesize
5-oxo-5,6-secocholestan-6-al-3ꢀ-(9-oxononanoate) (9-ON-secoA) as a white solid (111.3
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mg, 25%) or (33.2 mg, 6%). Data for 9-ON-secoA: Rf = 0.3 (EtOAc/n-hexane (1:3)); H
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NMR (400 MHz, CDCl3) ꢂ: 9.76 (t, J=2.0 Hz, 1H, CHO-9), 9.61 (s, 1H, CHO-7), 5.38 (s,
1H, H-3), 3.06 (dd, J=14.4, 4.4 Hz, 1H, H-4); 13C NMR (400 MHz, CDCl3) ꢂ: 216.08 (C-5),
202.84 (C-9’), 202.64 (C-7), 172.84 (C-1’), 73.14 (C-3), 56.12, 54.13, 52.34, 44.04, 43.84,
43.26, 42.54, 42.11, 39.85, 39.46, 35.97, 35.74, 34.80, 34.45, 34.38, 28.94, 28.92, 28.79,
28.71, 28.00, 27.82, 25.31, 25.15, 24.79, 23.74, 23.07, 22.79, 22.54, 21.98, 18.56, 17.64;
DART-TOF/MS (positive) calcd for C36H61O5 m/z [M+H]+ 573.4519, found 573.4410.
9-ON-secoA (69.7 mg, 0.12 mmol) was used to synthesize
5ꢀ-hydroxyl-B-norcholestane-6ꢀ-carboxaldehyde-3ꢀ-(9-oxononanoate) (9-ON-secoB) as a
white solid (21.9 mg, 32%) as described above. Data for 9-ON-secoB: Rf = 0.4
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(EtOAc/n-hexane (1:4)); H NMR (400 MHz, CDCl3) ꢂ: 9.76 (t, J=1.6 Hz, 1H, CHO-9’),
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9.68 (d, J=2.8 Hz, 1H, CHO-7), 5.13 (m, 1H, H-3), 2.50 (s, 1H, OH-5); C NMR (400
MHz, CDCl3) ꢂ: 203.84 (C-9’), 202.71 (C-7), 172.35 (C-1’), 83.65 (C-5), 70.17, 64.55,
56.03, 55.68, 51.49, 45.61, 44.66, 43.82, 42.29, 39.65, 39.48, 39.31, 36.19, 35.60, 34.55,
28.92, 28.82, 28.33, 27.99, 24.86, 24.66, 23.82, 22.78, 22.54, 21.95, 21.57, 18.75, 18.28,
12.49; DART-TOF/MS (positive) calcd for C36H61O5 m/z [M+H]+ 573.4519, found
573.4607.
Preparation of 9-oxononanoyl cholesterol. The authentic 9-oxononanoly cholesterol was
prepared as previously reported [20].
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Preparation of human LDL sample
This study was approved by the University of Shizuoka Ethical Committee. Ten ml
each of whole blood from healthy volunteers (n=6) was obtained in a 15 ml centrifugation
tube containing antioxidant (0.5 mg of 2,6-di-tert-butyl-4-methylphenol). The blood
samples were placed for 60 min at room temperature, and then centrifuged at 3000 rpm for
10 min to obtain the serum fraction. LDL (d 1.006–1.063 g/ml) was isolated by sequential
density ultracentrifugation from serum, dialyzed against 0.15M NaCl containing 1 mM
EDTA, and stored at 4°C. For lipid extractions, the LDL (50 μg/ml protein; 1 ml) was
mixed vigorously with 2 ml chloroform-methanol (2:1) for 1 min. After centrifugation at
3,000 rpm for 10 min, the organic phase was separated, washed with water twice, and
evaporated to dryness in vacuo. The residue was analyzed for the detection of secosterol-A,
secosterol-B, C16:0-secoA, C16:0-secoB, 9-ONC, 9-ON-secoA and 9-ON-secoB
(structures, see Fig. 1) after derivatization with DH as described in our previous study [16,
19, 21]. Briefly, aliquots of extracted samples from the human LDL were dissolved in 100
μl of acetonitrile containing 0.5 mg/ml DH and 0.1 mg/ml p-toluensulfonic acid, and
derivatized for 4 h at room temperature in darkness. The derivatized mixture was
evaporated to dryness in vacuo, and the residue was finally dissolved in 1 ml acetonitrile.
Then the DH derivatives of secosterols were analyzed by LC-ESI-MS/MS as described
below. Under these conditions, the recovery rates of these compounds added into 1 ml of
LDL (50 μg/ml protein) were more than 90%.
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LC-ESI-MS/MS analyses
LC-ESI-MS/MS was performed on an Agilent 1200 series HPLC system using a
TSK-GEL ODS-100V column (3 ꢀm, 150 × 2.0 mm, TOSOH, Tokyo, Japan) and an
Agilent G6410B triple quadrupole tandem mass spectrometer with an electrospray
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ionization device running in positive ionization mode. The detector conditions were as
follows: capillary voltage at 4000 V, source temperature at 300°C, drying gas flow at 7
l/min, nebulizer gas at 20 psi, and fragmentor at 200 V. The detection of compounds was
performed using the multiple reaction monitoring (MRM) mode. The ion transitions were
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monitored
for
secosterol-A-DH
and
-B-DH
(m/z
666.4>630.4,
365.3),
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3,4-13C-secosterol-A-DH and -B-DH (m/z 668.4>632.4, 236.0), C16:0-secoA-DH and
B-DH (m/z 904.7>630.2, 365.2), 9-ON-secoA-bisDH and B-bisDH (m/z 534.5>630.2,
365.2), 9-ONC-DH (m/z 788.2>420.0, 369.2), C18:1-CE (m/z 668.6>369.3, 206.5),
C18:2-CE (m/z 666.7>369.3, 206.0), C16:0-CE (m/z 642.5>369.3, 607.6), and cholesterol
(m/z 369.0>161.2, 147.1). For the analyses of DH derivatives, solvent A was 0.1% formic
acid in water and solvent B was 0.1% formic acid in acetonitrile. For the analyses of
unoxidized cholesterol and cholesteryl esters, solvent A was 10 mM ammonium formate
and solvent B was acetonitrile containing 30% isopropanol. Samples were separated using a
linear gradient of 70% solvent A and 30% solvent B to 100% solvent B in 20 min and with
100% solvent B for an additional 40 min. The flow rate was 0.2 ml/min. The level of
compounds were determined using 3,4-13C-secosterol-B as the internal standard.
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Assay for cytotoxicity
We have recently reported that secosterol-A, and -B, and their keto alcohol and
acid derivatives induce cell death in several human cultured cells including acute
promyelocytic leukemia HL-60, acute monocytic leukemia THP-1, acute T cell leukemia
Jurkat, histiocytic lymphoma U937, lung carcinoma A549, umbilical vein cell EA.hy926
and rat pheochromocytoma PC-12 [29]. These reports indicated that HL-60 cells are
relatively susceptible to secosterol-induced cell death compared to other cell type used in
the experiment. For these reasons, we evaluated the cytotoxic effects of secosterols in
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HL-60 cells (purchased from RIKEN Cell Bank Ibaraki, Japan), which were maintained in
10% FBS/RPMI 1640 supplemented with 50 units/ml penicillin and 50 μg/ml streptomycin
and were grown in an atmosphere of 95% air and 5% CO2 at 37ºC. The alamarBlue assay
was carried out for quantitative analysis of cell viability. After culturing, cells were seeded
in a 96-well plate, and then incubated with compounds at 37ºC for 24 h. Ten μl of an
alamarBlue solution was added to each well, and the fluorescence was measured using an
excitation at 560 nm and emission at 590 nm after incubation at 37ºC for 2–3 h in a
humidified CO2 incubator, according to the manufacturer’s direction. The obtained values
were compared with each of the controls incubated with vehicle only.
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RESULTS
Ozonolysis products of cholesteryl esters
Three major cholesteryl esters in human LDL, cholesteryl palmitate (C16:0-CE),
cholesteryl oleate (C18:1-CE), and cholesteryl linoleate (C18:2-CE), were treated with
ozone as described in the Materials and Methods section, and the major ozonolysis products
were isolated and characterized by MS and NMR analysis (Fig. 1). The major reaction
products of C16:0-CE with ozone were identified as C16:0-secoA and C16:0-secoB. On the
other hand, when C18:2-CE and C18:1-CE were exposed to ozone, their primary
ozonolysis product was identified as 9-ONC and two other ozonolysis products
9-ON-secoA and 9-ON-secoB were also isolated and characterized. These results suggest
that ozonolysis of cholesteryl esters with saturated fatty acid results in the formation of
secosterol esters with an unchanged fatty acid (Fig. 1). However, the reaction of ozone with
cholesteryl esters containing unsaturated fatty acids (C18:2 and C18:1) first yielded
cholesteryl esters with 9-oxononanoic acid, which was formed by the ozonolysis of C18:2
and C18:1. This core-aldehyde 9-ONC was then further oxidized to form 9-ON-secoA and
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9-ON-secoB (Fig. 1).
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Development of LC-ESI-MS/MS analysis of DNSL derivative of 9-ONC and secosterol
esters
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Authentic standards of C16:0-secoA, B, 9-ON-secoA, B, and 9-ONC were
subjected to ESI-MS/MS analyses after derivatization with DH. Base ions of C16:0-secoA
(B)-DH, 9-ON-secoE-A (B)-bisDH, and 9-ONC-DH, were detected at m/z 788.2 (z=1),
534.5 (z=2), and 904.7 (z=1), respectively, and their MS/MS spectra are shown in Fig. 2. To
measure the endogenous levels of the ozonolysis products of cholesteryl esters, we
optimized the analytical conditions for their LC-ESI-MS/MS-MRM analyses as described
in the Materials and Methods section. 9-ON-secoA-bisDH, 9-ON-secoB-bisDH,
9-ONC-DH, C16:0-secoA-DH, and C16:0-secoB-DH were eluted at 29.8, 30.5, 41.4, 47.5,
and 48.4 minutes, respectively (Fig. 3A). Detection limits of these compounds were ~1
fmol. In addition, cholesterol, C18:2-CE, C18:1-CE, and C16:0-CE were eluted at 35.2,
44.7, 49.5, and 50.5 minutes, respectively (Fig. 3B).
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15
16
17
18
19
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21
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25
Detection of secosterol ester in human LDL
In order to determine the physiological levels of secosterols and their derivatives,
human LDLs were isolated from six healthy volunteers (3 males and 3 females, aged
24.5 4.7), and analyzed for secosterols and their esterified derivatives. As shown in Table 1,
9-ONC, secosterol-A and secosterol-B were detected in all tested human LDL samples
(n=6) at 182.7 75.8, 58.8 11.5, and 33.6 16.5 (pmol/mg LDL protein), respectively.
Furthermore, we also detected 9-ON-secoA and 9-ON-secoB in all tested human LDL
samples (n=6) at 16.5 5.4, and 11.3 3.9 (pmol/mg LDL protein), respectively (Table 1).
However C16:0-secoA and C16:0-secoB were below the detection limits of the assay.
11

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Levels of the cholesteryl esters C18:2-CE, C18:1-CE and C16:0-CE were at 2448 567,
540 279, and 48 24 nmol/mg LDL protein, respectively (Table 1). The level of cholesteryl
arachidonate (C20:4-CE) was determined to be 155 58.7 nmol/mg LDL protein in human
LDL (n=6). The secosterol esters derived from 5-OVA, another core-aldehyde derived
mainly from C20:4-CE, were also below the detection limit in LDL from human healthy
volunteers (data not shown). These results indicate that low concentrations of 9-ON-secoA
and 9-ON-secoB are constitutively present in healthy human plasma LDL particles. In order
to investigate the effects of ozone exposure on the concentrations of secosterols and their
esterified derivatives, the samples of isolated human LDL were treated with ozone, and
then analyzed for these compounds using our developed method. As shown in Fig. 4,
treatment of human LDL with ozone resulted in increased levels of C16:0-secoA, and -B,
which were below the detection limits in untreated LDL samples. The levels of 9-ONC
detected in untreated LDL samples were markedly decreased by treatment with ozone,
whereas the levels of 9-ON-secoA, but not 9-ON-secoB, were significantly increased in
ozonized LDL, suggesting that 9-ONC is oxidized by ozone to yield 9-ON-secoA.
Additionally, it was confirmed that secosterol-A, but not secosterol-B, was also
significantly increased in ozonized LDL. Both 9-ON-secoA and secosterol-A were
markedly increased (approximately 1200-fold) when the human LDL samples were
exposed to ozone under the present experimental conditions.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Cytotoxic effect of secosterol esters on HL-60 cells
Secosterol-A and -B have been reported to be cytotoxic against several culture cell
lines [7, 10-12, 29]. Here we evaluated the cytotoxic effects of secosterol esters on human
promyelocytic leukemia HL-60 cells. As shown in Fig. 5, HL-60 cells viabilities were
decreased in a dose- and time-dependent manner by the treatment with 9-ON-secoA and
12

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9-ON-secoB, but not by the treatment with their parent compounds 9-ONC, C18:2-CE and
C18:1-CE. The cytotoxic effects of 9-ON-secoA and -B were as strong as those of the
unesterified secosterol-A and -B. When HL-60 cells were treated for 72 hours, IC50 values
of secosterol-A and -B were 3.8 and 6.9 μM, respectively, whereas the IC50s for
9-ON-secoA and -B were 4.0 and 4.5 μM, respectively. These results suggest that the
aldehyde group formed by the oxidation of the 5,6 double bond on cholesterol is an
important determinant of their cytotoxic activities.
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4
5
6
7
8
9
Discussion
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18
19
20
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22
23
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25
In this study, we reported for the first time the presence of 9-ON-secoA and -B in
human LDL, which are probably generated by oxidation of cholesterol esters (C18:1-CE
and C18:2-CE). The detection of these compounds was performed using a highly sensitive
and specific method developed in our previous study [16], which was based on
derivatization with DH and detection by LC-ESI-MS/MS. The levels of 9-ON-secoA and
-B in human LDL were 16.5 5.4, and 11.3 3.9 (pmol/mg protein), which corresponded to
20-50% of the unesterified forms of secosterol-A and -B, respectively, detected in human
LDL. The level of 9-ON-seco-A was markedly increased in vitro by the ozonolysis of
C18:1-CE and C18:2-CE, which are two major cholesterol esters found in human LDL.
In the present study, we detected 9-ON-secoA and -B in human LDL isolated from
healthy volunteers (n=6) at 16.5 and 11.3 pmol/mg LDL protein, respectively. These were
9.0 and 6.2% of 9-ONC, and 0.00055% and 0.00038% of the total amount of unoxidized
cholesteryl oleate and cholesteryl linoleate in LDL, respectively. Secosterol-A and -B were
also detected in the same samples at 55.8 and 33.6 pmol/mg LDL, respectively, which were
0.0021% and 0.0012% of unoxidized cholesterol. Free cholesterol seems to be 4–5-fold
more readily oxidized to form secosterols than cholesteryl oleate and cholesteryl linoleate.
13

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This might be due to the fact that cholesterol esters are present in the inner hydrophobic
core of LDL particles whereas free cholesterol is present in the outer membrane. Further
analyses are required to determine the pathological levels of secosterol esters in
atherosclerotic plaques or the brain tissues of dementia patients.
3
4
5
The levels of three major cholesteryl esters, cholesteryl linoleate, oleate, and
palmitate, in human plasma (n=6) were 2448 567, 540 279, and 48 24 nmol/mg LDL
protein, respectively (Table 1). 9-ON-secoA and -B detected in human LDL are oxidized
products of the two most abundant cholesteryl esters in LDL, cholesteryl linoleate and
oleate. On the other hand, C16:0-secoA and C16:0-secoB formed by the ozonolysis of
cholesteryl palmitate, were below the detection limits in human LDL isolated from healthy
volunteers, which may be due to the fact that the amount of their respective parent
compound was limited. However these derivatives may be formed and increased by
inflammatory stimuli. Further studies are needed to analyze the association of these
secosterol esters in human tissue with development of atherosclerosis and Alzheimer and
Parkinson diseases.
6
7
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9
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25
With regard to the origin of secosterols in human LDL, it is still controversial
whether ozone is necessary for the oxidative cleavage of the ꢃ5,6 double bond of the steroid
structure to form secosterol-A, although secosterol-B was shown to be formed not only by
aldolization of secosterol-A, but also by an ozone-independent pathway via Hock cleavage
of 5ꢁ-hydroperoxycholesterol [22]. Uemi et al. suggested that secosterol-A is also formed
via unstable cholesterol dioxetane in an ozone-independent pathway by the reaction of
cholesterol with singlet oxygen [23]. On the other hand, Wentworth et al. previously
demonstrated using an aqueous buffer system that secosterol-A was only formed by the
reaction of cholesterol with ozone and not from the reaction of other various ROS such as
singlet oxygen, superoxide anion, and hydroxyl radicals [24]. We have also previously
14

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reported that secosterol-A was formed in vivo by a reaction with an ozone-like oxidant
generated with PMA-activated neutrophils through an MPO-dependent mechanism [16].
Moreover, in the present study, we observed that secosterol-A, C16:0-secoA, and
9-ON-secoA, but not their aldolization products, secosterol-B, C16:0-secoB, and
9-ON-secoB, were markedly increased in human LDL treated with ozone (Fig. 4). Further
studies are required to explore the mechanism for formation and elimination of free and
esterified secosterols.
3
4
5
6
7
8
We also found that 9-ON-secoA and -B, but not 9-ONC, exert cytotoxic effects on
HL-60 cells, which suggests that the aldehyde groups formed by the oxidative cleavage of
the ꢃ5,6 double bond of the steroid structure is an important determinant of their cytotoxic
activities (Fig. 5). Their cytotoxicities were as potent as unesterified secosterol-A and -B.
Additionally, other reports also suggest these aldehyde groups are involved in
proatherogenic roles such as protein aggregation, fibrilization, and induction of scavenger
receptor (CD36 and SR-A) expression [9, 13-15, 25-28]. Alteration of their molecular
polarities may cause translocation from the inner part to the outer surface of the LDL
particle which could trigger several bioactivities.
9
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12
13
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18
19
20
21
22
23
24
25
In this study, we have reported for the first time the detection of the novel
cytotoxic secosterol esters 9-ON-secoA and -B in human LDL, which are formed by
oxidation of cholesteryl esters. As the aldehyde group in these secosterol derivatives is
important for the execution of their bioactivities, further studies are needed to determine the
association of the levels of free and esterified secosterols with human diseases caused by
chronic inflammation such as cancer, diabetes, atherosclerosis and neurodegenerative
disorders.
15

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Acknowledgments
This work was supported in part by a JSPS KAKENHI (24680075 to NM,
3
4
24700838 to ST and 24300257 to HO).
5
6
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2013.
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Figure legends
Figure 1. Chemical structures and oxidation pathway of cholesteryl esters and their
ozonolysis products.
5
6
Figure 2. MS/MS spectra of ozonolysis products formed from cholesteryl esters. Authentic
standards of C16:0-secoA, -B, 9-ON-secoA, -B, and 9-ONC were derivatized with DH,
then infused to ESI-MS/MS analyses. Collision-induced dissociation voltages for
C16:0-secoA, B-DH, 9-ON-secoA, B-bisDH, and 9-ONC-DH were set at 29, 29, 13, 13,
and 29V, respectively.
7
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9
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13
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16
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Figure 3. Typical chromatograms obtained by LC-ESI-MS/MS analyses. DH derivatives of
secosterol-A, -B, 9-ON-secoA, -B, 9-ONC, C16:0-secoA, and -B (A) and cholesterol,
C18:2-CE, C18:1-CE, and C16:0-CE (B) were subjected LC-ESI-MS/MS-MRM analyses.
The ion transitions were monitored for secosterol-A-DH and -B-DH (m/z 666.4>630.4),
9-ON-secoA-bisDH and B-bisDH (m/z 534.5>630.2), 9-ONC-DH (m/z 788.2>369.2),
C16:0-secoA-DH and B-DH (m/z 904.7>630.2), cholesterol (m/z 369.0>147.1), C18:2-CE
(m/z 666.7>369.3), C18:1-CE (m/z 668.6>369.3), and C16:0-CE (m/z 642.5>369.3).
Figure 4. Effect of ozonolysis of human LDL on the formation of secosterol esters. Human
LDL (50 μg protein/ml obtained from subject No. 4 shown in Table 1) was treated with
(lower in each panels) or without (upper in each panels) ozone for 10 min in phosphate
buffer (pH 7.4). Extracted lipid fractions were derivatized with DH, then subjected to
LC-ESI-MS/MS. MRM chromatogram of 9-ON-secoA, B-bisDH (m/z 534.5>630.2);
C16:0-secoA, B-DH (m/z 904.7>630.2); secosterol-A, B-DH (m/z 666.4>630.4);
9-ONC-DH (m/z 788.2>369.2).
Figure 5. Cytotoxic effects of ozonolysis products of cholesteryl esters on HL-60 cells.
20

1
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3
4
5
6
7
8
9
HL-60 cells were treated with 9-ON-secoA (ꢅ), 9-ON-secoB (ꢆ), 9-ONC (Ⱦ), C18:1-CE
(ꢇ) and C18:2-CE (ꢈ) at indicated concentration for 24 h. Cell viabilities were determined
by alamarBlue assay. Values are shown as the mean SD (n=3).
Highlights
ꢀ Human LDL cholesteryl esters were treated with ozone and the products identified
ꢀ The products were also detected in human LDL samples by LC-ESI-MS/MS
ꢀ The products 9-oxononanoyl secosterol-A and -B were cytotoxic to HL-60 cells
ꢀ These products may play roles in the pathogenesis of inflammatory disorders.
21

Graphicalꢀabstract
Miyoshi N.ꢀetꢀal.
O3
C18:1ꢁcholesterol
Aldolization
O3
9ꢁONC
9ꢁONꢁsecoA
9ꢁONꢁsecoB
O3
C18:2ꢁcholesterol

Figure 1, Miyoshi et al.
Esterified with saturated fatty acid
H
H
O
H
O
O
Aldolization
O3
O
(CH2)14CH3
C16:0-cholesterol
C16:0-secoA
C16:0-secoB
Esterified with unsaturated fatty acid
H
H
H
O
O3
O
(CH2)7
(CH2)7CH3
C18:1-cholesterol
Aldolization
O3
H
9-ONC
9-ON-secoA
9-ON-secoB
H
H
O3
O
O
(CH2)7
(CH2)4CH3
C18:2-cholesterol

Figure 2, Miyoshi et al.
C16:0-secoA-DH
䢸䢵䢲䢰䢴
[M+H]+
䢵䢸䢶䢰䢺
䢴䢵䢷䢰䢺
䢳䢲䢲 䢴䢲䢲 䢵䢲䢲 䢶䢲䢲 䢷䢲䢲 䢸䢲䢲 䢹䢲䢲 䢺䢲䢲 䢻䢲䢲
C16:0-secoB-DH
䢸䢵䢲䢰䢲
[M+H]+
䢵䢸䢷䢰䢲
䢴䢵䢷䢰䢺
䢵䢻䢷䢰䢲
䢳䢲䢲 䢴䢲䢲 䢵䢲䢲 䢶䢲䢲 䢷䢲䢲 䢸䢲䢲 䢹䢲䢲 䢺䢲䢲 䢻䢲䢲
9-ON-secoA-bisDH
[M+2H]2+
䢶䢴䢲䢰䢳
䢸䢵䢲䢰䢶
䢷䢴䢷䢰䢶
䢳䢹䢲䢰䢴
䢵䢸䢷䢰䢶
䢴䢵䢷䢰䢻
䢳䢲䢲 䢴䢲䢲 䢵䢲䢲 䢶䢲䢲 䢷䢲䢲 䢸䢲䢲 䢹䢲䢲 䢺䢲䢲 䢻䢲䢲 䢳䢲䢲䢲
9-ON-secoB-bisDH
[M+2H]2+
䢸䢵䢲䢰䢷
䢶䢴䢲䢰䢴
䢷䢴䢷䢰䢴
䢳䢹䢲䢰䢳
䢴䢵䢸䢰䢳
䢵䢸䢷䢰䢶
䢴䢹䢺䢰䢲
䢳䢲䢲 䢴䢲䢲 䢵䢲䢲 䢶䢲䢲 䢷䢲䢲 䢸䢲䢲 䢹䢲䢲 䢺䢲䢲 䢻䢲䢲 䢳䢲䢲䢲
9-ONC-DH
䢵䢸䢻䢰䢴
[M+H]+
䢶䢴䢲䢰䢲
䢳䢸䢳䢰䢲
䢶䢲䢴䢰䢲
䢴䢶䢵䢰䢴
䢳䢲䢲
䢴䢲䢲
䢵䢲䢲
䢶䢲䢲
䢷䢲䢲
䢸䢲䢲
䢹䢲䢲
䢺䢲䢲

Figure 3, Miyoshi et al.
A
Secosterol-A, B-DH
9-ON-secoA, B-bisDH
C16:0-secoA, B-DH
9-ONC-DH
m/z 666.4>630.4
m/z 534.5>630.2
m/z 788.2>369.2
m/z 904.7>630.2
20
60
Time (min)
B
Cholesterol
C18:2-CE
C18:1-CE
m/z 369.0>147.1
m/z 666.7>369.3
C16:0-CE
m/z 668.6>369.3
m/z 642.5>369.3
20
60
Time (min)

Figure 4, Miyoshi et al.
Bis-DH derivatives of 9-ON-secoA and B
DH derivatives of C16:0-secoA and B
(m/z 534.5 > 630.2)
(m/z 904.7 > 630.2)
A
B
7E+01
2E+03
1E+03
0E+03
2E+03
1E+03
0E+03
4.7E+01
4.6E+01
4.5E+01
6E+01
ND
5E+01
O3 (-)
O3 (-)
A
A
2E+03
1E+03
0E+03
2E+03
1E+03
0E+03
O3 (+)
O3 (+)
20
40
Time (min)
60
20
40
60
Time (min)
DH derivatives of secosterol-A and -B
DH derivatives of 9-ONC
(m/z 666.4 > 630.4)
(m/z 788.2 > 369.2)
8E+01
7E+01
6E+01
5E+01
2E+05
1E+05
B
4E+02
A
2E+02
O3 (-)
O3 (-)
0E+05
2E+05
A
8E+01
7E+01
6E+01
5E+01
1E+05
0E+05
O3 (+)
O3 (+)
20
40
60
20
40
60
Time (min)
Time (min)

Figure 5, Miyoshi et al.
150
100
50
0
0
5
10
Concentration (µM)

Tableꢀ1.ꢀLevelsꢀofꢀoxidizedꢀandꢀunoxidizedꢀsteroidalꢀcompoundsꢀinꢀhumanꢀLDL.
SecosterolꢁAꢀSecosterolꢁBꢀ9ꢁONꢁsecoA9ꢁONꢁsecoB 9ꢁONC C16:0ꢁCE C18:2ꢁCEꢀ C18:1ꢁCEꢀCholesterol
SubjectꢀNo.ꢀ
(pmol/mgꢀLDLꢀprotein)
(nmol/mgꢀLDLꢀprotein)
1ꢀ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
53.2ꢀ ꢀ7.4ꢀ 48.0ꢀ ꢀ6.6ꢀ 18.6ꢀ 1.6
7.6ꢀ 1.8ꢀ 264ꢀ 64.7ꢀ77.5ꢀ 25.92179ꢀ ꢀ810ꢀ596ꢀ ꢀ307ꢀ2506 369
52.5ꢀ ꢀ15.1ꢀ 53.1ꢀ ꢀ16.4ꢀ 19.2ꢀ 13.6 10.4ꢀ 3.6ꢀ 288 7.7ꢀ 74.8ꢀ 27.53089ꢀ ꢀ463ꢀ736ꢀ ꢀ145ꢀ2780 480
55.5ꢀ ꢀ1.5ꢀ 42.8ꢀ ꢀ5.4ꢀ 18.0ꢀ 2.4 7.3ꢀ 1.4ꢀ 175 27.4ꢀ34.2ꢀ 2.6 2847ꢀ ꢀ207ꢀ928ꢀ ꢀ81ꢀꢀ 2617 308
62.6ꢀ ꢀ19.9ꢀ 18.5ꢀ ꢀ1.7ꢀ 11.8ꢀ 1.6 13.7ꢀ 0.4ꢀ 127 25.0ꢀ32.7ꢀ 1.9 1964ꢀ ꢀ152ꢀ296ꢀ ꢀ38ꢀꢀ 2278 171
72.9ꢀ ꢀ13.4ꢀ 21.9ꢀ ꢀ5.5ꢀ 12.9ꢀ 3.2 16.0ꢀ 1.4ꢀ 119 12.0ꢀ31.4ꢀ 0.8 2006ꢀ ꢀ3ꢀꢀ 270ꢀ ꢀ19ꢀꢀ 2998 187
56.4ꢀ ꢀ0.7ꢀ 17.3ꢀ ꢀ1.1ꢀ 18.6ꢀ 3.2 12.5ꢀ 5.0ꢀ 125 32.7ꢀ34.5ꢀ 0.9 2604ꢀ ꢀ640ꢀ418ꢀ ꢀ231ꢀ3123 186
Mean SDꢀofꢀtriplicatedꢀanalysesꢀareꢀshown.
ꢀ