A unique structural distribution pattern discovered for the cerebrosides from starfish Asterias amurensis

Article abstract of DOI:10.1016/j.carres.2018.12.013

Cerebroside is an important family of the mono-glycosylated ceramides involved in the larger family of glycosphingolipid and sulfatide. Cerebroside is synthesized from ceramide by the transfer of glucose from UDP-glucose, and degraded back to ceramide, which plays an important role at the epidermis protecting interior of the body as a barrier. Because cerebroside is regarded as the source molecule of ceramide and is amphiphilic in nature, cerebroside is considered valuable as the ingredient of cosmetic lotion. Various sources can be considered as raw material of cerebrosides. Starfish is considered as one of such potent source. However, the structure of the ceramide part of cerebroside is not fully investigated. Therefore, the individual structures of cerebroside molecules need to be identified including sphingosine and fatty acyl group composition to assess the potential of the molecule. We investigated and determined the structures of cerebrosides in starfish Asterias amurensis using LC-MS, GC-MS, tandem mass spectrometry (MS/MS), and ¹H NMR. We also discovered a characteristic structure distribution that was divided into three major groups: 1) a group composed of a relatively long sphingosine (C22) and a short length of fatty acyl group (less than C16), 2) a group composed of a typical C18 sphingosine and long fatty acyl groups (greater than C23), and 3) a group composed of C18 sphingosine and fatty acyl groups with their length less than C18. The calculated Log P values of cerebrosides ranging from 9 to 11 covered about 80% of the molecules that were in the range of those used in cosmetics, thus showing the potential usefulness of starfish Asterias amurensis as a source of raw material for cerebrosides.

Full text of DOI:10.1016/j.carres.2018.12.013

Accepted Manuscript
A unique structural distribution pattern discovered for the cerebrosides from starfish
Asterias amurensis
Ryosuke Yamaguchi, Yoshimi Kanie, Osamu Kanie, Yoshitaka Shimizu
PII:
S0008-6215(18)30687-6
https://doi.org/10.1016/j.carres.2018.12.013
CAR 7654
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 24 November 2018
Revised Date: 20 December 2018
Accepted Date: 20 December 2018
Please cite this article as: R. Yamaguchi, Y. Kanie, O. Kanie, Y. Shimizu, A unique structural distribution
pattern discovered for the cerebrosides from starfish Asterias amurensis, Carbohydrate Research
(2019), doi: https://doi.org/10.1016/j.carres.2018.12.013.
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ACCEPTED MANUSCRIPT

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A unique structural distribution pattern discovered for the
cerebrosides from starfish Asterias amurensis
Ryosuke Yamaguchi, Yoshimi Kanie, Osamu Kanie*, Yoshitaka Shimizu
Department of Applied Biochemistry, Tokai University, 4-1-1 Kitakaname, Hiratsuka,
Kanagawa 259-1292 Japan
e-mail: 7bjam041@mail.u-tokai.ac.jp (RY), yoskanie@tsc.u-tokai.ac.jp (YK),
kanie@tokai-u.jp (OK), shimizuy@tsc.u-tokai.ac.jp (YS)
*Corresponding author information:
Osamu Kanie, Professor, Tokai University
e-mail: kanie@tokai-u.jp
Affiliation and address: Department of Applied Biochemistry, Tokai University
4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292 Japan
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ABSTRACT:
Cerebroside is an important family of the mono-glycosylated ceramides involved in
the larger family of glycosphingolipid and sulfatide. Cerebroside is synthesized from
ceramide by the transfer of glucose from UDP-glucose, and degraded back to ceramide,
which plays an important role at the epidermis protecting interior of the body as a
barrier. Because cerebroside is regarded as the source molecule of ceramide and is
amphiphilic in nature, cerebroside is considered valuable as the ingredient of cosmetic
lotion.
Various sources can be considered as raw material of cerebrosides. Starfish is
considered as one of such potent source. However, the structure of the ceramide part of
cerebroside is not fully investigated. Therefore, the individual structures of cerebroside
molecules need to be identified including sphingosine and fatty acyl group composition
to assess the potential of the molecule.
We investigated and determined the structures of cerebrosides in starfish Asterias
amurensis using LC-MS, GC-MS, tandem mass spectrometry (MS/MS), and 1H NMR.
We also discovered a characteristic structure distribution that was divided into three
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major groups: 1) a group composed of a relatively long sphingosine (C22) and a short
length of fatty acyl group (less than C16), 2) a group composed of a typical C18
sphingosine and long fatty acyl groups (greater than C23), and 3) a group composed of
C18 sphingosine and fatty acyl groups with their length less than C18. The calculated
Log P values of cerebrosides ranging from 9 to 11 covered about 80% of the molecules
that were in the range of those used in cosmetics, thus showing the potential usefulness
of starfish Asterias amurensis as a source of raw material for cerebrosides.
Keywords
Cerebroside, starfish Asterias amurensis, structure distribution, structure diversity,
structure determination
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1. Introduction
Ceramide is a class of molecules that consists of a sphingosine and fatty acyl group
linked through an amide bond, which is modified posttranslationally, generally creating
diverse molecular species including glycosphingolipids and sphingomyelin. Among
them, the mono-glycosylated ceramides are called cerebrosides.
Human epidermis consists of five distinctive layers: outermost stratum corneum,
stratum lucidum, stratum granulosum, stratum spinosum, and innermost stratum basale.
Each layer displays one of the sequential differentiation stages of keratinocytes, the
major cell type in the epidermis. It was evidenced that ceramide accounts for over 50%
of the subcutaneous fat tissue in stratum corneum. It maintains a permeability barrier
against transepidermal water loss, exogenous molecules, and pathogen invasion. [1–4]
Cerebroside is located in lamellar granular cells in stratum granulosum and may be a
source of ceramide in the stratum corneum. [5–8] Because of the amphiphilic nature of
cerebrosides, as a result of a hydrophilic sugar moiety and hydrophobic ceramide, they
could be a beneficial ingredient for retaining moisture on the skin surface, which is why
cosmetic lotions containing cerebrosides have been commercialized. Moreover,
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cerebroside is considered a therapeutic agent against atopic disease in food markets
because of the improved skin barrier function provided by dietary cerebroside. [9–13] A
variety of biological functions are attributed to cerebrosides, such as anti-tumor activity
in early stage colon cancer, anti-inflammatory activity, plant-growth regulation, as well
as antimicrobial activity. [14–22]
Despite the wide distribution of cerebrosides in animal and plant tissues, bovine
brain and horse spinal cord were the major source. Other sources of cerebrosides are
grains, beans, fungi, and especially wheat that contains a relatively high content (20-40
mg/100 g). Although cerebrosides of plant and fungi origin have been considered as a
substitute of those from animal sources, the amount of cerebrosides in plants and fungi
is small, and purification is expensive. [23, 24]
On the other hand, starfish Asterias amurensis, inhabiting the North Pacific ocean,
contains more cerebrosides in their organs (820 mg/100 g) than the above-mentioned
plant and fungi species. [25] Outbreak of starfish causes severe damage to the fishery
and aquaculture ground for benthic shellfish. More than 15,000 tons of starfish Asterias
amurensis are being scrapped in Hokkaido, Japan every year. The continuous
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extermination of starfish is becoming a burden to fisherman. The wasted starfish could
have become a potent source of cerebrosides. It was recently reported that a specific
cerebroside from the starfish could induce ceramide synthesis in human epidermis. [26]
Cerebroside consists of heterogeneous sphingoid base and fatty acyl groups, and the
individual structures have been analyzed separately. [27–29] Several reports have been
published regarding individual structures of major components from starfish by using
NMR and MS. [30-33]
We thought it important to identify the individual structures of cerebrosides and
their composition in order to use the cerebrosides from the starfish as a cosmetic
material. In this study, we determined the structures and composition of cerebrosides in
starfish Asterias amurensis using HPLC-MS, MS/MS, NMR, and GC-MS, and found a
characteristic structure distribution.
2. Experimental
2.1. Materials
The starfish Asterias amurensis was collected in 2013 off the coast of
Saruhutsumura, Hokkaido, Japan. Sep-Pak C18 was purchased from Waters (MA,
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USA). All solvents and reagents were reagent grade or LC grade. Milli-Q water was
used.
2.2. LC-ELSD and LC-MS analysis of cerebrosides
Reversed-phase, high-performance liquid chromatography (HPLC) was carried out
on DP-8020 (Tosoh, Tokyo, Japan) with Inertsil ODS-3 column (4.6 × 250 mm,
GL-science, Tokyo, Japan) with Sedex 75 evaporative light scattering detector (ELSD)
(SEDERE, Alfortville, France). Methanol/water (98:2, v/v) was used as mobile phase at
flow rate 0.9 mL/min. Sample was separated at room temperature.
HPLC-chromatogram was acquired and processed with Chromato-PRO (Runtime
Instruments, Tokyo, Japan).
LC-MS was carried on LCMS-IT-TOF system (Shimadzu, Kyoto, Japan) with
above-mentioned conditions. The solvent flow was split down under 0.2 mL/min before
introduction into electrospray ionization interface. The mass spectrometric parameters
were as follows: polarity: positive ion mode, ion source gas (nitrogen): 1.5 L/min, dry
gas pressure: 155 kPa, interface voltage: 4.5 kV, curved dissolved line temperature and
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heat block temperature: 200 °C, detector voltage: 1.68 kV and scan range: 100 to 2000
m/z. Data were analyzed using LabSolution Workstation (Shimadzu, Kyoto, Japan).
2.3. ESI-IT-MS and MS/MS analysis of cerebrosides
ESI-MS and MS/MS analyses were carried out on AmaZon equipped with
electrospray ionization interface. (Bruker Daltonics, Bremen, Germany) The sample
was directly infused into electrospray ionization mass spectrometers. The mass
spectrometric parameters were as follows: polarity: positive ion mode, ion source gas
(nitrogen) pressure: 8.0 psi, dry gas: 5.0 L/min, dry temperature: 220 °C, and scan
range: 100 to 2000 m/z. Sample was dissolved in methanol. In MS/MS experiment, the
precursor isolation window was set to ±2 Da and helium was used as the collision gas.
Operation was carried on trap Control software and data were acquired and processed
with Compass DataAnalysis (Bruker Daltonics GmbH, Bremen, Germany).
2.4. NMR of a glucoside
1H and 13C spectra were obtained in CDCl3 (tetramethylsilane as an internal
standard, 0.00 ppm) using an Avance 500 MHz NMR Spectrometer. (Bruker Biospin,
MA, USA) NMR spectra were acquired and processed with Topspin workstation.
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2.5. Gas chromatography and GC-MS analysis of fatty acid methyl ester
GC analysis was carried out on GC-2014 gas liquid chromatograph (Shimadzu,
Kyoto, Japan) equipped with a column of G-300 (40 m, 1.2 mm ID, 2.0 µm, CERI,
Tokyo, Japan) on FID detection. The column temperature was initially set at 180 °C for
3 min, then increased to 230 °C at a rate of 15 °C/min and maintained at 230 °C for 30
min. The injector and detector temperature were set at 270 and 300 °C, respectively.
Helium was used as a carrier gas at a flow rate of 47 cm/sec. Then, 1 µL of sample was
injected. Chromatogram was recorded on C-R7A plus. (Shimadzu, Kyoto, Japan)
GC-MS analysis was carried out on QP-5050 gas chromatograph-mass spectrometer
(Shimadzu, Kyoto, Japan) with a column of Omegawax (30 m, 0.25 mm ID, 0.25 µm,
Restec Co., PA, USA) on EI mode. The column temperature was initially set at 50 °C
for 5 min, then increased to 250 °C at a rate of 8 °C/min and maintained at 250 °C for
30 min. The injector and MS interface temperature were set at 250°C. Helium was used
as a carrier gas at a flow rate of 52.1 cm/sec. Then, 1 µL of sample was injected on split
mode. EI mass spectra were acquired and processed using LabSolution Workstation.
(Shimadzu, Kyoto, Japan)
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2.6. GC- MS analysis of ozonolysis products
Same equipment was used as FAME (fatty acid methyl ester). Ozonolysis products
were dissolved in 100 µL of n-propanol for GC-MS analysis. The same GC-MS
conditions were used as described above except that the column temperature was
initially set at 80 °C for 5 min, then increased to 200 °C at a rate of 6 °C/min and
maintained at 200 °C for 30 min. Helium was used as a carrier gas at a flow rate of 44.7
cm/sec and 1 µL of sample was injected on split mode.
2.7. MALDI-TOF-MS analysis of glucosylsphingosine (GlcSph)
MALDI-TOF-MS analyses were carried out on AutoFlex. (Bruker Daltonics,
Bremen, Germany) The sample dissolved in methanol was spotted on a target plate with
dihydroxybenzoic acid (DHB). The mass spectrometric parameters were as follows:
polarity: positive ion mode, Nd/YAG laser: 355 nm, and scan range: 600 to 1000 m/z.
MS spectra were acquired with FlexControl and processed with FlexAnalysis (Bruker
Daltonics, Bremen, Germany).
2.8. Extraction of cerebrosides
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Total lipids were extracted following the Bligh and Dyer method [34] from whole
bodies of starfish, organs, and ovaries, separately. Obtained chloroform layer was
evaporated and dried under vacuum. Total lipid was separated by using silica gel
chromatography. Neutral lipids were eluted with chloroform/methanol/water (90:10:0.1,
v/v/v), and concentrated under vacuum. The major constituents in the neutral lipid
fraction were cerebrosides.
2.9. Oxidative degradation of cerebrosides and acetylation
Cerebrosides (98 mg wet weight) were suspended in 300 µL ethanol/water (1:2).
Then, 600 µL of 5% sodium hypochlorite (NaClO) was added and stirred vigorously at
room temperature for 1 h.
Then n-Octanol (150 µL) and formic acid (15 µL) were added sequentially to a mixture
and stirred at 4 °C for 1 h. The water layer was collected after centrifugation of the
reaction mixture for 5 min at 3000 rpm. Supernatant was applied onto Sep Pak-C18
(150 mg/4.0 mL) and eluted by 10 mL of water and 10 mL of methanol. The eluent was
evaporated and dried under vacuum. The residue was dissolved in pyridine (1 mL) and
added acetic anhydride (2 mL). The mixture was stirred at 50 °C for 1 h. Excess amount
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of reagent was decomposed by adding methanol at 4 °C. The reaction mixture was
evaporated and dried under vacuum to give the crude product, which was purified on a
silica gel column (hexane:ethyl acetate, 1:2, v/v) to yield acetylated glycoside.
2.10. Fatty acid analysis by acidic methanolysis
Cerebroside (65 mg/mL) in ethanol (1 mL) was dried under nitrogen gas and 5%
HCl-MeOH (1 mL) was added. The mixture was heated at 100 °C for 30 min. After
cooling to room temperature, the reaction mixture was extracted by hexane twice, and
the hexane layer was subsequently washed with water. The hexane layer was collected
and dried to yield a fatty acid methyl ester.
2.11. Preparation of GlcSph by alkaline hydrolysis
Cerebroside (10 mg) was dissolved in 1N KOH in n-butanol (1 mL), and the
mixture was heated at 100 °C for 4 h to obtain GlcSph. The mixture was then
neutralized by using 2N HCl. The lyso-form was extracted by chloroform/methanol (2:1,
v/v) eight times. Compound was further purified on a silica gel column using
chloroform/methanol/water (65:25:4, v/v/v) as an eluent.
2.12. Ozonolysis of double bonds
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Sphingosines and fatty acids were fractionated by RP-HPLC prior to the ozonolysis
of the sample. Each fraction was dissolved in chloroform and cooled at -70 °C, and then
ozone gas was passed through the medium at room temperature for 30 min.
Immediately after the reaction, 2.5 mL of NaBH4 in methanol (13 mg/2.5 mL) was
added. The reaction mixture was allowed to stand at room temperature for 60 min with
occasional stirring and extracted twice with chloroform/methanol/water (3:48:47, v/v/v)
after the addition of 1.5 mL of water. The obtained products were evaporated to
dryness.
2.13. Calculation of Log P values
Log P values were calculated by using a software MarvinSketch (ver 18.13.0,
ChemAxon; https://chemaxon.com/products/marvin).
3. Results
3.1. Structures of cerebrosides from whole body of starfish
The structures of cerebrosides obtained from the dry powder of the whole body of
starfish were estimated by MS/MS analysis after separation on reversed-phase HPLC.
Estimated molecular species such as sugar moiety, fatty acyl group and glycosyl
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sphingosine were further assessed individually after various derivatizations, according
to the reported method. The position of double bonds in ceramide was determined by
ozonolysis following GC-MS (Fig. 1).
3.1.1. Composition analysis of cerebrosides in starfish, Asterias amurensis by LC-
ELSD and LC-MS
The detected m/z values and determined structure are summarized in Table 1. More
than 23 peaks were observed by HPLC and some of them consisted of several
components (Fig. 2). The observed ions appeared in the range of m/z 700 to 900 as
sodium adducted form in ESI-MS. The most dominant ion was observed at m/z 792.5
(25.4 min, peak 14). Another peak (peak 15, 26.6 min), which also has the same m/z
value (m/z 792.5), was a structural isomer of compound in peak 14. A structural isomer
was also detected for the second dominant component at m/z 778.5 as it appeared as two
peaks, and they were considered to be positional isomers of the double bond in fatty
acyl group. The ion (m/z 778.5) was assumed to consist of one carbonless ceramide with
m/z 792.5, which could be determined by MS/MS. The third dominant component was
the ion at m/z 844.6. It seemed at this point that the cerebrosides obtained from Asterias
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amurensis formed two groups of molecules. One was observed around m/z 792.5 and
the other around m/z 844.6.
3.1.2. Structure analysis of cerebroside by MS/MS
The typical MS/MS spectrum of cerebroside containing br18:3/h16:0 ceramide:
N-[(2S,3R,4E,8E,10E)-1,3-dihydroxy-9-methyl-4,8,10-octadecatriene-2-yl]-2’-hydroxyl
hexadecanamide at m/z 748.6 is shown in figure 3. The cleavage of glyosidic linkage
and amide linkage of fatty acyl group were clearly observed at m/z 586.6 and 494.3,
respectively in MS/MS analysis. The mass numbers of constituting sphingosine and
fatty acyl group could be assumed by the calculation of fragment ions mass number.
Other examples were shown in figure S1. After carrying out MS/MS experiments on
individual cerebrosides, GlcSph was found to consist of d18:1, d18:2, d18:3, br18:3,
d22:1, d22:2, t22:1 and t22:2. Fatty acyl group-oriented fragment ion was observed that
underwent cleavage of glycosidic linkage and C2–C3 in sphingosine, i.e. m/z 320.3 in
figure 3 [33], which provided information regarding their composition in addition to
those obtained from GC as discussed below.
3.1.3. Determination of monosaccharide and its anomeric configuration
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We determined a sugar structure and an anomeric configuration, as it has been
reported that two cerebrosides consisting of β-glucose and β-galactose as sugar moiety
in starfish. [35] Cerebrosides were subjected to oxidative degradation using NaClO
followed by acetylation of existing hydroxyl groups, which were assigned by 1H NMR
analysis. The method of oxidative degradation was simple and easily accessed due to
the use of commercial bleach in aqueous condition. [36] NaClO treatment of
cerebrosides gave glycan nitrile compound as a result of releasing most of the ceramide
with retained glycosidic linkage, as well as anomericity. Moreover, the acetylation of
hydroxyl groups is useful to obtain easily assignable NMR spectrum due to the
ester-shift of ring protons. The 1H NMR spectrum showed a set of distinct signals for
H1 and H4 observed at δ4.73 ppm (doublet, J1,2 = 7.9 Hz) and δ5.11 ppm (triplet, J3,4
J4,5 = 9.9 Hz), respectively. COSY- and HSQC-NMR were also examined for
confirmation (Fig. S2). As a result, it was identified that the constituting carbohydrate
was glucose with β-configuration. GalCer has been reported to exist in four starfish
species only. [35] There has not been a report describing the presence of GalCer in
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Asterias amurensis. Although we cannot deny the possibility, the major component of
the cerebroside in Asterias amurensis is GlcCer.
3.1.4. Analysis of fatty acyl composition by GC-FID and GC-MS
Next, constituting fatty acyl structures and their composition were determined
based on GC-MS and GC-FID after methanolysis. Nineteen fatty acid methyl esters
(FAMEs), having hydroxyl group at C2 in a chain length varying from C14 to C25,
were observed. Among them, h16:0 (35.0%) and h24:1 (19.2%) were the major
constituents (Table 2, Fig. S3). The overall components of fatty acids were in good
agreement with the reported data, which described the composition of cerebrosides in
the spermatozoa of the starfish, Asterias amurensis. [27] The major fatty acids detected
were h14:0, h15:0, h16:0, h18:0, h23:1, and h24:1 over 5% content. This was somewhat
different in that the content of h14:0 in the whole body was relatively smaller than that
in the spermatozoa. The mono-unsaturated acids were only found in the longer chain
acids (C22-C25). The fatty acids with intermediate chain length (C19-C21) were
scarcely detected. The determined composition of fatty acids by GC-FID was consistent
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with the composition of the fatty acyl groups calculated from its component
cerebrosides in Table 1.
3.1.5. Structure analysis of glucosyl sphingosine by MS
Furthermore, we carried out MS analysis of GlcSph obtained by alkaline hydrolysis
of cerebrosides to clarify sphingosine structure. [37] GlcSph signals were observed as
protonated and sodiated ions by MALDI-TOF-MS (Fig. S4). Table 3 shows the
observed mass numbers and calculated long-chain base structures. We determined 8
sphingosines, most of which contained one to three double bonds. The major
constituents were found to be d18:2, d18:3, br18:3 and d22:1, which was similar to
results from spermatozoa of the starfish. [27] However, t22:1 was observed in very low
intensity, which might be caused by the low ionization efficiency in MALDI-TOF-MS.
Odd numbered long-chain might contain a branching methyl group.
3.1.6. Determination of double bond position in ceramide by GC-MS
The position of a double bond was partially confirmed by ozonolysis. [38] The long
chain alcohol obtained after ozonolysis and subsequent reduction, was analyzed by
GC-MS. The major alcohols observed were from C8 to C13 (Fig. S5A). The position of
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a double bond could be assumed from these alcohols. The peak 14 and 15 (d22:2/h16:0,
m/z 792.5) in figure 2 after ozonolysis and reduction yielded 1-nonanol and 1-tridecanol
Products in each GC-MS chromatogram suggested that the presence of d22:2∆4,13 and
d22:2∆4,9 in peak 14 and 15, respectively. (Fig. S5B,C) Although it was not possible to
determine the trans- and cis-isomeric structures, the related compound d22:2/h16:0 has
been reported, in the same species, consisting of a sphingosine with a trans-double bond
at C4 and a cis-double bond at C9 or C13. [33] It was also reported that a ceramide
br18:3/h24:1 contains trans-double bonds at C4, 8, and 10 and an acyl group with a
cis-double bond at C15. In our case, the position of double bond in fatty acid was
determined as follows. The ozonolysis and reduction of a sample forming a peak of
HPLC (peak 20, m/z = 844.8) of cerebroside yielded 1-octanol and 1-nonanol. MS/MS
analysis of this fraction (Fig. S1G) shows C18 sphingosine having three double bonds
(d18:3) where terminal one is located at C10, which indicates the ozonolysis may
provide octanol. Therefore, the 1-nonanol observed after ozonlysis belongs to a C24
fatty acid (h24:1), and its position is C15. The trans- and cis-isomeric structure was
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assigned according to the reference 33. The results were well-matched to previously
reported data. [27]
3.2. Composition of cerebrosides from a whole body of starfish, viscera, and gonad
The relative amounts of each component of cerebrosides from a whole body, gonad,
and viscera of starfish Asterias amurensis are shown in Table 1. These results were
obtained by HPLC with evaporative light-scattering detector (ELSD), which had a
linear response within the experimental order of magnitude. [23] Peak 10 in Figure 2
consisted of cerebroside having branch type br18:3 sphingosine and h18:0 fatty acyl
group (br18:3/h18:0) and other minor unidentified components. The relative
composition of peak 10 was observed to be higher in gonad and viscera, than in the
whole body. Major components observed in the whole body were found in both viscera
and gonad as well (Fig. S6). The most dominant constituent was cerebroside containing
d22:2 sphingosine and h16:0 fatty acyl group (d22:2/h16:0) with 24.5%, 23.7% and
21.6% in the whole body, gonad, and viscera, respectively. It seemed that the observed
structure distribution was not organ-specific. However, the components that varied site
specificity were observed and the compositions of the molecules containing ceramide
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d22:1/16:0 and d18:1/h23:1 were increased in gonad compared to those molecules in
viscera. Meanwhile the compositions of the molecules containing ceramide d18:3/h14:0,
d18:2/h22:1 and d18:3/h24:1 were increased in viscera compared to in the gonad.
Moreover, the molecule containing h14:0 fatty acyl group was scarcely observed in
gonad. Although the information of ceramide synthase (CerS) about starfish was
unknown, it was suggested that several kinds of CerSs existed and the expression level
of the specific CerS was different in each site, as well as in mammals. [39–41]
4. Discussion
We determined the structures of cerebrosides in whole starfish using LC-MS and
MS/MS combined with other established methods, such as GC-MS, GC-FID, and NMR
in order to reveal an overview of the structure distribution of cerebroside. We found the
critical structure preferences in distribution of molecules forming three groups, that 1)
the length of the fatty acid was limited to less than C16 in case of the relatively long
sphingosine such as C22:1, 2) the relatively long fatty acid more than C23 was observed
in the case of the typical sphingosine length at C18, and 3) a combination of shorter
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sphingosine and fatty acids (Fig. 4). Interestingly, a combination of long sphingosine
and fatty acids could not be observed. It was considered that the structure preference in
this starfish might depend on 1) the total lipid contents, 2) substrate specificity of
ceramide-glucosyltransferase, and/or 3) substrate specificity of ceramide synthase. Fatty
acid composition in internal organs has been reported to consist of C18:0, C20:1∆5,
C20:4∆5,8,11,14 and C20:5∆5,8,11,14,17 were major components. It was also reported that
these lipids accounted for more than 67% and 73% in viscera and gonad, respectively.
[29] These distributions of fatty acids were clearly different from the distribution of the
fatty acid in cerebroside, leaving the possibilities of other two. Unfortunately, the
substrate specificity of ceramide-glucosyltransferase or ceramide synthase of starfish is
not known. Although we could refer to the information obtained from research in other
sources, such as mammals, not much information has been reported regarding their
substrate specificity. The chirality of amide nitrogen-attached carbon atom is important
and the hydrophobicity of substrate must be preferred in the ceramide recognition for
ceramide–glucosyl transferase [42], although it seems the lengths of both chains are not
related to the transfer reaction.
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Ceramides are synthesized by (1) de novo pathway and (2) salvage pathway. The
latter involves the sphingomyelin hydrolysis generating phosphocholine and ceramide
by sphingomyelinase. [40, 41, 43] The distribution of ceramide structures in other
sphingolipids, such as sphingomyelin, could be used as references as well. However,
there has been no report about the composition of sphingomyelins, because of lesser
content compared to cerebroside in the starfish. Six ceramide synthases with different
substrate specificities have been reported in mammals. [39, 40] These ceramide
synthases, probably representing a family of isozymes, combine a fatty acyl-CoAs and a
sphinganine to form a dihydroceramide. [41] Fumonisin B1 that consists of aminoalkyl
backbone with anionic tricarballylic side chains has been reported as an inhibitor, which
competes with the sphingosine binding site of (dihydro)ceramide synthase and
interferes with utilization of the co-substrate fatty acyl-CoA. [44] The steric hindrance
in the enzyme catalytic site by a long acyl chain of sphingosine with an unsaturated
bond might be problematic, such fatty acyl-CoA may not be able to approach in the
binding site. Alternatively, the long fatty acyl group might fit in the pocket in the case
of C18 sphingosine. We expect further research about substrate specificity in ceramide
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synthases. As it was a characteristic feature in the structure distribution that the
ceramide structures formed three groups, these characters of cerebrosides should be
important in composing liposomes and micelles in cosmetics. [45]
The moisture preservation effect of cerebroside has been recognized in cosmetics.
Cerebroside is more hydrophilic compared with ceramide, due to the added five
hydroxyl groups in sugar moiety. We compared log P value of cerebroside in starfish to
those in others to evaluate the moisture preservation effect. Log P is employed as the
numerical value of the partition coefficient between n-octanol and water, which
indicates the hydrophobic nature of cerebroside. [46] Less hydrophobic compounds
shows more affinity toward water and will keep moisture at the skin surface. The log P
values of cerebrosides in starfish were distributed in the range from 6.61 to 11.43, and
nearly 80 % of cerebrosides were accounted in the range from 9 to 11 (Fig. 4). On the
other hand, the log P values of ceramides in human skin surface were distributed from
9.99 to 22.74. Furthermore, the log P values of major cerebrosides derived from plants,
those were also considered as the potent ingredients in cosmetics, were found as
follows; 7.86 (d18:2/h16:0) in soybean, 8.75 (d18:2/h18:0) in konjak and 9.64
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(d18:2/h20:0) in rice and corn. Although the extra hydroxyl group in a fatty acyl group
in starfish cerebrosides makes log P value lower, those values in starfish were observed
higher than those in plant because of the longer chain of sphingoid base. These feature
of starfish cerebrosides may be considered as potent as other cerebrosides obtained from
other sources for cosmetics to attract more moisture.
Moreover, a cerebroside (br18:2/d16:0) with branched sphingosine was known to
show antimicrobial activity and suppression of melanin formation in melanoma cell. [15,
47, 48] The starfish cerebrosides contains the branching structure, therefore it could be
advantageous for skin protection.
5. Conclusion
Cerebrosides were isolated from gonad, viscera, and whole body of the starfish
Asterias amurensis by means of solvent extraction, silica gel chromatography, and
reversed-phase high-performance liquid chromatography. The structures of cerebrosides
were finally characterized based on tandem mass spectrometry (overall structure) by
combining partial structures obtained by GC-MS (fatty acid), and MS and GC after
ozonolysis (unsaturation position).
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Cerebroside structure distribution was observed in ceramide whose fatty acyl
groups extended from C14 to C24, containing α-hydroxyl group with saturated or
monounsaturated form and sphingosine were C18 and C22 with unsaturated form.
These were divided into three groups. This information of structure distribution will be
useful in composing liposomes and micelles. [49, 50] We also calculated the log P
values of those cerebrosides and found them suitable for moisturizing effects in
cosmetics.
Acknowledgment
The authors acknowledge Professor Koretaro Takahashi of Kitami Institute of
Technology for his valuable discussion. The authors also thank Dr. Yoshiki Oda of
Research Promotion Division in Tokai University for NMR analysis.
This research is partly supported by Research and Study Program of Tokai University
General Research Organization and also Matching Planner Program from Japan Science
and Technology Agency, JST.
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