Lipids (2015) 50:987–996
DOI 10.1007/s11745-015-4054-4
ORIGINAL ARTICLE
Milk Phospholipids Enhance Lymphatic Absorption of Dietary
Sphingomyelin in Lymph-Cannulated Rats
Masashi Morifuji1 · Seiichiro Higashi1 · Chisato Oba1 · Satomi Ichikawa1 ·
Keiko Kawahata1 · Taketo Yamaji1 · Hiroyuki Itoh1 · Yuki Manabe2 ·
Tatsuya Sugawara2 Received: 5 June 2015 / Accepted: 9 July 2015 / Published online: 2 August 2015
© AOCS 2015
Abstract Supplementation with sphingomyelin has been
reported to have beneficial effects on disease prevention
and health maintenance. However, compared with glycerolipids, intact sphingomyelin and ceramides are poorly
absorbed. Therefore, if the bioavailability of dietary sphingomyelin is increased, then the dose administered can be
reduced. This study was designed to identify molecular
species of ceramide in rat lymph after the ingestion of
milk sphingomyelin, and to compare the effect of purified sphingomyelin with milk phospholipids concentrate
(MPL, 185 mg sphingomyelin/g) on lymphatic absorption of milk sphingomyelin. Lymph was collected hourly
for 6 h from lymph-cannulated rats (n = 8/group) after
the administration of a control emulsion (triolein, bovine
serum albumin, and sodium taurocholate), a sphingomyelin
emulsion (control + purified sphingomyelin), or a MPL
emulsion (control + MPL). Molecular species of ceramide
in lymph were analyzed using high-performance liquid
chromatography-tandem mass spectrometry (HPLC–MS/
MS). Molecular species of ceramide, containing not only
d18:1, but also d17:1 and d16:1 sphingosine with 16:0,
22:0, 23:0, and 24:0 fatty acids (specific to milk sphingomyelin), were increased in rat lymph after the administration of milk sphingomyelin. Their molecular species were
similar to those of dietary milk sphingomyelin. Recovery
of ceramide moieties from dietary sphingomyelin was
* Masashi Morifuji
[email protected]
1
Food Science Research Labs, Meiji Co., Ltd., 540 Naruda,
Odawara‑shi, Kanagawa 250‑0862, Japan
2
Division of Applied Biosciences, Graduate School
of Agriculture, Kyoto University, Kitashirakawaoiwakecho,
Sakyo‑ku, Kyoto, Kyoto 606‑8502, Japan
1.28- to 1.80-fold significantly higher in the MPL group
than in the sphingomyelin group. Our results demonstrated
that dietary sphingomyelin from milk was transported to
lymph as molecular species of ceramide hydrolyzed from
milk sphingomyelin and co-ingestion of sphingomyelin
with glycerophospholipids enhanced the bioavailability of
dietary sphingomyelin.
Keywords Bioavailability · Sphingomyelin · Ceramides ·
Lymph · Rats · Mass spectrometry
Abbreviations
HPLC–MS/MS
High-performance liquid chromatography–tandem mass spectrometry
MPLMilk phospholipids
MRMMultiple-reaction-monitoring
Introduction
Sphingomyelin is not only a constituent of cell membranes,
but also a dietary component. Sphingomyelin is most abundant in eggs, meat, milk, and fish [1–3]. Supplementation with
sphingomyelin has been reported to have beneficial effects
on disease prevention and health maintenance, such as lowering serum cholesterol [4], preventing colon cancer [5, 6],
improvement of skin barrier function [7–9]. Administration of
sphingomyelin has also been shown to reduce the number of
aberrant colonic crypt foci and to decrease the proportion of
carcinomas to adenomas induced by 1,2-dimethylhydrazine
in mice [5, 6]. Furthermore, dietary sphingomyelin extracted
from bovine milk increased hydration of the stratum corneum
and reduced transepidermal water loss in mice [7–9].
Sphingolipid-hydrolyzing intestinal enzymes cleave
dietary sphingolipids to their component sphingoid bases,
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Lipids (2015) 50:987–996
Fig. 1 Typical chemical
structure of sphingomyelin from
milk, and their degradation
products: a N-(tricosanoyl)hexadecasphing-4-enine1-phosphocholine (sphingomyelin, d16:1/23:0-SM), b
N-(tricosanoyl)-hexadecasphing-4-enine (ceramide,
d16:1/23:0), c hexadecasphing4-enine (sphingosine, d16:1)
fatty acid moieties, and polar head groups before uptake
by intestinal mucosal cells (Fig. 1). Most absorbed sphingoid bases are rapidly metabolized to palmitic acid, but
part of them are reincorporated into sphingolipids and
transported into the systemic circulation through lymph
[10, 11]. According to Nilsson [10], after the administration of sphingosine-labeled sphingomyelin to rats, a small
amount of the radiolabeled ceramides were found in lymph
fluid. A recent study demonstrated that molecular species of ceramide, such as N-palmitoyl-4,8-sphingadienine (d18:2/16:0), and N-tricosanoyl-4,8-sphingadienine
(d18:2/23:0), were identified in rat lymph after they were
treated with maize glucosylceramide [12]. Sphingomyelin from milk comprise various molecular species which
contain different length carbon chains of the sphingoid
bases and fatty acids [13]. However, it remains unclear
what molecular species of ceramide were absorbed into rat
lymph after the ingestion of dietary sphingomyelin.
Studies in rats have shown that intact sphingomyelin
and ceramides are poorly absorbed, because the sequential breakdown to fatty acids and sphingoid bases, which is
necessary for absorption, is slow and incomplete compared
with the rapid digestion of glycerolipids. Therefore, if the
bioavailability of dietary sphingomyelin is increased, then
the dose administered can be reduced. Bovine milk lipids
contain approximately 0.5–1 % of phospholipids, which
are comprised mainly of sphingomyelin and phosphatidylcholine [2]. Plasma concentrations of poorly absorbed
drugs have been shown to increase when formulated with
sphingomyelin [14, 15]. Therefore, we hypothesized that
co-ingestion of dietary sphingomyelin and other glycerophospholipids from milk, such as milk phospholipids
(MPL) concentrate, might improve the bioavailability of
dietary sphingomyelin compared with sphingomyelin consumed in its purified from. The aim of the present study
was to identify molecular species of ceramide in rat lymph
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after the ingestion of milk sphingomyelin, and to compare
the effect of purified sphingomyelin with MPL on the lymphatic absorption of dietary sphingomyelin in rats.
Materials and Methods
Animals
Twenty-four male Sprague–Dawley rats weighing 300–
320 g (Nippon SLC Inc., Shizuoka, Japan) were used in
this study. All rats were multiply housed (three rats per
plastic cage) in a temperature- and humidity-controlled
room (22 ± 1 °C and 50 ± 10 % relative humidity, respectively) with a 12-h light–dark cycle. All animal experiments in this study were approved by Meiji Co., Ltd. Institutional Animal Care and Use Committee, and performed
in accordance with the Guiding Principles for the Care and
Use of Laboratory Animals (2014_3871_0038).
Collection of Lymph
After an acclimatization period of 1 week on standard commercial laboratory rodent chow (MF; Oriental Yeast, Tokyo,
Japan) with free access to distilled water, the rats were
anesthetized using sodium pentobarbital (64.8 mg/mL,
Table 1 Composition of test emulsion (/3 mL)
Control Sphingomyelin MPL
Triolein (mg)
Albumin from bovine serum (mg)
Sodium taurocholate (mg)
Milk phospholipids (MPL) (mg)
200
50
200
–
195
50
200
–
177.05
50
200
27
Sphingomyelin (mg)
–
5
–
Lipids (2015) 50:987–996
Somno-pentyl, Kyoritsu Seiyaku Corp., Tokyo, Japan). A
cannula (SV35, Natsume Seisakusyo Co. Ltd., Tokyo, Japan)
was inserted into their left thoracic channel to collect lymphatic fluid, and a catheter (SP-55, Natsume Seisakusyo Co.
Ltd., Tokyo, Japan) was inserted into their stomach. After
surgery, rats were placed in an individual restraining cage
in a warm recovery room. A physiological solution containing 139 mmol/L glucose and 85 mmol/L NaCl was continuously infused through a stomach cannula overnight at a rate
of 3 mL/h; and the same solution was also provided as drinking water. The morning after lymph was collected over 1 h
for use as a blank control, the rats were infused with 3 mL
of an emulsion, as a single bolus, through a stomach catheter (n = 8/group). The compositions of the test solutions are
shown in Table 1. All three solutions (control, sphingomyelin, and MPL) contained the same components in the control solution: triolein (Wako Pure Chemicals Industries, Ltd.,
Osaka, Japan), bovine serum albumin (Wako Pure Chemicals
Industries, Ltd.), and sodium taurocholate (Wako Pure Chemicals Industries, Ltd.). The purified milk (purity > 98 %)
sphingomyelin solution (Nagara Science Co. Ltd., Gifu,
Japan) also contained sphingomyelin. The MPL solution
(phospholipid concentrate 700) (Fonterra Co. Ltd., Auckland,
New Zealand) contained MPL comprised of 185 mg/g sphingomyelin, 193 mg/g phosphatidylcholine, 137 mg/g phosphatidylethanolamine, and 58 mg/g phosphatidylinositol. The
fatty acid composition of the MPL comprised 5.5 % myristic
acid (14:0), 19.3 % palmitic acid (16:0), 11.1 % stearic acid
(18:0), 39.1 % oleic acid (18:1), 5.3 % linoleic acid (18:2),
and 1.8 % linolenic acid (18:3). Fat and sphingomyelin contents in all test solutions were adjusted to 200 mg/3 mL, and
5 mg/3 mL, respectively. The compositions of test emulsions
were prepared by ultrasonication. The infusion of the prepared emulsions into rats was immediately followed by the
infusion of a glucose-NaCl solution. Lymph was collected in
a 10 % (w/v) disodium EDTA-containing tube (100 μL/h) for
analysis during the following intervals after the test-oil infusion: −1 to 0, 0–1, 1–2, 2–3, 3–4, 4–5, and 5–6 h. After the
collected lymph volume was measured, lymph was stored at
−80 °C until analysis.
Analysis of the Ceramide Moieties in Milk
Sphingomyelin
Analysis of the ceramide moieties in milk sphingomyelin
was performed following the method of Blaas et al. [16].
MPL and purified sphingomyelin were suspended in 20 μL
of 10 % Triton X-100 solution and 80 μL of 200 mM Tris–
HCl (pH 7.4). After sonication for 1 min and incubation
for 1 min at 37 °C, 0.08 mL of Tris–HCl solution (200 mM
Tris, 5 mM MgCl2, pH 7.4) and 10 μL of 1 U sphingomyelinase (S9396, Sigma-Aldrich Corp., Saint-Louis, MO,
989
USA) were added. This mixture was incubated for 2 h at
37 °C in a water bath. The reaction was stopped by the
addition of 1.5 mL of chloroform/methanol (2:1, v/v) and
0.2 mL of purified water. After vortexing and centrifugation, the upper aqueous phase was removed. The lower
organic phase was dried at 37 °C. After the residue had
been dissolved in methanol, the solution was analyzed
using high-performance liquid chromatography-tandem
mass spectrometry (HPLC–MS/MS).
Lipid Extraction from Lymph
Lipids were extracted from each sample of lymph with 4
volumes of chloroform/methanol (2:1, v/v). After centrifugation, the lower layer was collected, dried, and resolved
using methanol and analyzed using HPLC–MS/MS. The
recovery percentage of each ceramide from lymph samples
ranged from 99.4 to 100.4 %.
Analysis of the Molecular Species of Ceramide
Molecular species of ceramide were identified, and quantified using a HPLC–MS/MS system (Quattro premier XE,
Waters Corporation, Milford, MA, USA). All analyses were
performed on a 2.1 × 100 mm column with a particle size
of 1.7 µm (ACQUITY UPLC BEH C18, Waters Corporation). Mobile phase A consisted of 5 mM ammonium acetate
in 95 % methanol, while mobile phase B consisted of 5 mM
ammonium acetate in acetonitrile. The initial eluent composition was 100 % A, followed by an increase to 100 %
B for 30 min, 100 % B for 2 min, and then a reduction to
0 % A for 3 min. Total run time was 35 min. The eluent
flow was 0.4 mL/min and the column temperature was set
at 40 °C. Analytes were detected using electrospray ionization in the positive mode. Multiple-reaction-monitoring
(MRM) was performed using characteristic fragmentation
ions (d18:1/14:0, m/z = 492.4/264.3; d18:1/16:0, m/z = 520.4/264.3; d18:1/18:0, m/z = 548.5/264.3; d18:1/18:1,
m/z = 546.5/264.3; d18:1/20:0, m/z = 576.5/264.3; d18:1/
22:0, m/z = 604.6/264.3; d18:1/23:0, m/z = 618.6/264.3;
d18:1/24:0, m/z = 632.6/264.3; d18:1/24:1, m/z = 630.6/
264.3; d18:1/25:0, m/z = 646.6/264.3; d18:1/26:0, m/z = 660.6/264.3; d17:1/16:0, m/z = 506.4/250.3; d17:1/22:0,
m/z = 590.6/250.3; d17:1/23:0, m/z = 604.6/250.3; d17:1/
24:0, m/z = 618.6/250.3; d16:1/16:0, m/z = 492.4/236.3; d16:1/
22:0, m/z = 576.5/236.3; d16:1/23:0, m/z = 590.6/236.3;
d16:1/24:0, m/z = 604.6/236.3). The parameters for HPLC–
MS/MS analysis of ceramides were as follows: capillary voltage, 3000 V; source temperature, 120 °C; desolvation temperature, 400 °C; desolvation gas flow, 850 L/h; cone gas flow,
50 L/h; cone voltage, 40 V; and collision energy, 30 eV. Each
individual peak was identified by comparing the retention
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time with that of analytical standard of ceramide (d18:1/16:0,
d18:1/18:0, d18:1/20:0, d18:1/22:0, d18:1/24:0, Avanti Polar
lipids, Inc., Alabaster, AL, USA), or purified non-hydroxy
fatty acid ceramides (C2137, Sigma-Aldrich Corp.). Contents
of d18:1/23:0 ceramide molecular species were calculated
using a d18:1/24:0 ceramide standard. A ceramide containing
d16:1 sphingosine was quantified using ceramide standards
with d18:1 sphingosine. The recovery of ceramides in lymph
(0–6 h) was calculated for the sum of lymph ceramide contents, from which was subtracted the baseline levels (−1–0 h)
at each interval. The recovery rates of ceramide moieties from
dietary sphingomyelin were calculated by dividing the recovery of ceramides in lymph by the lipid contents administered
via dietary sphingomyelin.
Lipids (2015) 50:987–996
Statistical Analysis
All data are presented as mean ± standard error (SEM).
Data were analyzed by one-way ANOVA with post hoc
analysis being performed using Tukey’s honestly significant difference test. Comparisons between the MPL and
the sphingomyelin group were via Student’s t-tests (SPSS
ver.22.0, SPSS, IL, USA). Differences between groups
were considered to be significant at P < 0.05.
Results
Change in Lymph Volume and Lymph Triacylglycerol,
Phospholipids Content
Analysis of Triacylglycerol and Phospholipids in Lymph
Triacylglycerol levels in lymph were measured using commercial kits (Triglyceride E-Test Wako, Wako Pure Chemicals Industries., Ltd.). The levels of choline-derived phospholipids, mainly phosphatidylcholine and sphingomyelin,
in lymph were measured using enzymatic kits (Phospholipid C-Test Wako, Wako Pure Chemicals Industries., Ltd.).
Figure 2 shows the change in lymph volume and lymph
triacylglycerol, phospholipids content.
Lymph volume was not different among the three
groups. Both lymph triacylglycerol and phospholipids
levels peaked at 1–2 h in all groups but were not different
among the three groups.
Fig. 2 Change of lymph volume, triacylglycerol, and phospholipids content in rat lymph. The values are shown as means ± SEM (n = 8/group)
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Fig. 3 Analysis of the ceramide moieties of milk sphingomyelin
used in the experiment: a chromatogram of purified sphingomyelin,
b chromatogram of milk phospholipids (MPL), and c peak percentage
area of ceramide moieties of milk sphingomyelin. Arrow heads show
the identified peaks of ceramides
Analysis of Ceramide Moieties in Milk Sphingomyelin
before administration of MPL, whereas only trace levels of
ceramide containing d16:1 and/or, d17:1 sphingosine were
identified (Fig. 4a). Administration of MPL significantly
increased the levels of molecular species of ceramide containing d16:1, d17:1, and d18:1 sphingosine with either
16:0, 22:0, 23:0, or 24:0 fatty acids in rat lymph (Fig. 4b).
However, ceramide containing sphingosine with oleic acid
(18:1) was only detected at trace levels after the administration of MPL.
The composition of molecular species of ceramide in rat
lymph 0–6 h after the ingestion of either sphingomyelin or
MPL is shown in Fig. 4c. The ceramides in rat lymph, as a
percentage of the area of molecular species, was not different between the sphingomyelin and MPL groups.
The analytical results for ceramide moieties in milk sphingomyelin are shown in Fig. 3.
Molecular species of ceramide containing d18:1 sphingosine with 16:0, 22:0, 23:0, and 24:0 fatty acids were
mainly identified in both sphingomyelin and MPL hydrolyzed by sphingomyelinase. Furthermore, ceramides containing d16:1 and/or d17:1 sphingosine with 16:0, 22:0,
23:0, and 24:0 fatty acids were also detected in both hydrolyzed sphingomyelin and MPL (Fig. 3a, b). Compositions
of the molecular species of ceramide in purified sphingomyelin were similar to those in MPL. Sphingomyelin and
MPL were comprised of 84 % of molecular species of ceramide containing d16:1 and d18:1 sphingosine with 16:0,
22:0, 23:0, and 24:0 fatty acids. Therefore, after the rats
ingested milk sphingomyelin, we focused on the recovery
of these molecular species of ceramide.
Identification of Molecular Species of Ceramide in Rat
Lymph Before and After the Administration of MPL
Figure 4a, b shows a chromatogram depicting the molecular species of ceramide in rat lymph before and after the
administration of MPL. Molecular species of ceramide
containing d18:1 sphingosine with either 16:0, 22:0, 23:0,
24:0, or 24:1 fatty acids were mainly detected in rat lymph
Change in Molecular Species of Ceramide Containing
d18:1 Sphingosine in Rat Lymph
Figure 5 shows the change in molecular species of ceramide containing d18:1 sphingosine in rat lymph after
administration of the control, the MPL, or sphingomyelin
solution. Similar to the results for triacylglycerol in lymph,
the molecular species of ceramide in lymph reached maximum levels at 1–2 h in all groups,
The ingestion of sphingomyelin significantly increased
d18:1/16:0 and d18:1/23:0 ceramide contents at the 0‒1,
1‒2, and 2‒3 h intervals compared with the control. A
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Lipids (2015) 50:987–996
Fig. 4 Typical chromatogram of the molecular species of ceramide in rat lymph a before and b after the administration of MPL.
Arrow heads show the identified peaks of ceramides. c The composi-
tion of the molecular species of ceramide in rat lymph 0–6 h after
the ingestion of sphingomyelin and MPL. The values are shown as
means ± SEM (n = 8/group)
significant increase in d18:1/22:0 and d18:1/24:0 ceramide
contents were observed in the sphingomyelin group at
0‒1 h, compared with the control group.
Ceramide levels of d18:1/16:0, and d18:1/23:0 were
significantly higher in the MPL group than in the control
group at every time interval assessed. The MPL solution
significantly increased d18:1/22:0 and d18:1/24:0 ceramide
levels at the 0‒1, 1‒2, and 2‒3 h intervals, compared with
the control solution.
group than in the control group at every time interval
assessed.
Change in Molecular Species of Ceramide Containing
d16:1 Sphingosine in Rat Lymph
Figure 6 shows the change in molecular species of ceramide containing d16:1 sphingosine in rat lymph.
The ingestion of sphingomyelin significantly increased
d16:1/16:0 and d16:1/23:0 ceramide contents at the 0‒1,
1‒2, 2‒3, and 4‒5 h intervals, compared with the control.
A significant increase in the d16:1/22:0 ceramide level was
observed in the sphingomyelin group at the 0‒1, 1‒2, and
2‒3 h intervals compared with the control group. A significantly higher level of d16:1/24:0 ceramide was observed
in the sphingomyelin group at the 0‒1, 1‒2, 2‒3, 3‒4, and
4‒5 h intervals, compared with the control group.
Ceramide levels of d16:1/16:0, d16:1/22:0, d16:1/23:0,
and d16:1/24:0 were significantly higher in the MPL
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Recovery Rate of Ceramide Moieties from Dietary
Sphingomyelin
Data for the recovered ceramide moieties from dietary
sphingomyelin are summarized in Table 2. The recovery
of molecular species of ceramide was significantly higher
in the MPL group than in the sphingomyelin group for the
following: d18:1/16:0, d18:1/22:0, d18:1/23:0, d18:1/24:0,
d16:1/16:0, d16:1/22:0, d16:1/23:0, and d16:1/24:0. Compared with sphingomyelin, the MPL administered significantly increased the recovery rate of ceramide moieties
from sphingomyelin containing d18:1/16:0, d18:1/22:0,
d18:1/23:0, d18:1/24:0, d16:1/16:0, d16:1/22:0, d16:1/23:0,
and d16:1/24:0.
Discussion
Using HPLC–MS/MS analysis, we identified the molecular
species of ceramide in rat lymph after the ingestion of milk
sphingomyelin. Molecular species of ceramide, containing
not only d18:1 sphingosine, but also d17:1 and d16:1 sphingosines, which are specific to milk sphingomyelin, were
Lipids (2015) 50:987–996
993
Fig. 5 Changes in the molecular species of ceramide containing d18:1 sphingosine with 16:0, 22:0, 23:0, and 24:0 fatty acids in rat lymph. The
values are shown as mean ± SEM (n = 8/group). Means without a common letter are significantly different, P < 0.05
detected in rat lymph after the ingestion of milk sphingomyelin. Interestingly, dietary milk sphingomyelin was transported to lymph as ceramide structures and their molecular
species were similar to those of dietary milk sphingomyelin.
Furthermore, co-ingestion of dietary sphingomyelin with
glycerophospholipids, as compared to purified sphingomyelin, significantly enhanced the recovery of ceramide moieties from dietary sphingomyelin in rat lymph.
The most prevalent long-chain base of mammalian
sphingolipids is d18:1 sphingosine, while milk sphingomyelin is comprised of molecular species of sphingomyelin
containing not only d18:1, but also d17:1 and d16:1 sphingosine [13]. After the ingestion of MPL, ceramides containing d18:1/16:0, d18:1/22:0, d18:1/23:0, and d18:1/24:0
were mainly increased, whereas other molecular species
of ceramide with d18:1 sphingosine were only slightly
increased (Fig. 4). Furthermore, after the administration
of MPL, it was interesting to observe a great increase in
molecular species of ceramide containing d17:1 and d16:1
sphingosines with 16:0, 22:0, 23:0, and 24:0 fatty acids in
rat lymph. However, a ceramide containing sphingosine
with oleic acid (18:1) was detected at a trace level after the
ingestion of milk sphingomyelin, even though an excess
amount of triolein was co-ingested with milk sphingomyelin. Thus, dietary sphingomyelin were transported to
lymph as ceramide structures, and their molecular species
were similar to those found after hydrolysis of milk sphingomyelin. After the administration of sphingosine-labeled
sphingomyelin to rats, a small amount of radiolabeled ceramide was found in the lymph fluid [10]. Research as early
as 1968 indicated that after the feeding of sphingosinelabeled ceramide, a small amount of radioactive ceramide
was detected in its intact form in intestinal tissue [10]. We
speculated it is therefore likely that hydrolyzed dietary
sphingomyelin is directly absorbed in its intact form into
lymph, but further studies are needed to clarify the potential underlying mechanism by which this occurs.
A ceramide containing d18:1 sphingosine was increased
in the lymph of the control group, even though dietary
sphingomyelin was not administered to these rats. Dietary fats are hydrolyzed in the lumen of the intestine and
absorbed by enterocytes. Lipids are subsequently resynthesized in the endoplasmic reticulum and are secreted with
chylomicrons. Chylomicrons are comprised of 86‒92 %
triacylglycerol and 6‒8 % phospholipid. Chylomicron
phospholipids contain mainly phosphatidylcholine and
have smaller amounts of sphingomyelin and ceramides [17,
18]. Therefore, one possible explanation for this increase in
d18:1 sphingosine in lymph could be that lymph chylomicrons are secreted accompanying the intestinal absorption
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Lipids (2015) 50:987–996
Fig. 6 Changes in the molecular species of ceramide containing d16:1 sphingosine with 16:0, 22:0, 23:0, and 24:0 fatty acids in rat lymph. The
values are shown as means ± SEM (n = 8/group). Means without a common letter are significantly different, P < 0.05
Table 2 Recovery rate of ceramide moieties from dietary sphingomyelin
Lipid content administered
(nmol/3 mL sample)
Recovery in lymph (nmol)
Sphingomyelin
Sphingomyelin
MPL
Recovery rate (%)*
MPL
P value
Sphingomyelin
MPL
P value
<0.001
d18:1/16:0
583
580
7.65 ± 0.44
11.43 ± 0.63
<0.001
1.31 ± 0.08
1.97 ± 0.10
d18:1/22:0
287
255
2.33 ± 0.27
3.72 ± 0.54
0.042
0.81 ± 0.09
1.46 ± 0.20
0.017
d18:1/23:0
482
406
14.06 ± 0.77
19.34 ± 1.91
0.026
2.85 ± 0.16
4.65 ± 0.43
0.003
d18:1/24:0
295
259
9.25 ± 0.48
14.76 ± 1.30
0.001
2.99 ± 0.16
5.46 ± 0.45
<0.001
d16:1/16:0
109
145
4.54 ± 0.15
7.74 ± 0.57
0.001
4.15 ± 0.14
5.34 ± 0.37
0.015
d16:1/22:0
329
358
2.25 ± 0.16
3.32 ± 0.28
0.007
0.68 ± 0.05
0.92 ± 0.07
0.025
d16:1/23:0
573
554
17.15 ± 1.00
22.43 ± 1.84
0.028
2.99 ± 0.17
4.05 ± 0.31
0.017
d16:1/24:0
335
348
14.04 ± 0.93
18.90 ± 1.68
0.028
4.20 ± 0.28
5.43 ± 0.45
0.049
Values are means ± SEM (n = 8/group)
* The recovery rates of ceramide moieties from dietary sphingomyelin were calculated by dividing the recovery of ceramides in lymph by the
lipid contents administered via dietary sphingomyelin
of dietary fats, resulting in an increase in these endogenous
ceramides in rat lymph.
We focused on the molecular species of ceramide
containing d16:1 sphingosine in order to be able to distinguish the effects of endogenous ceramide contents
in lymph. After treatment with the control solution, the
molecular species of ceramide with d16:1 sphingosine
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were unchanged. The ingestion of purified sphingomyelin significantly increased the recovery of ceramides
with a d16:1 sphingosine compared with the control.
Thus, ceramides containing sphingosine d16:1, which
is specific to bovine milk, were suitable markers for the
evaluation of the lymphatic absorption of dietary milk
sphingomyelin.
Lipids (2015) 50:987–996
This study provided a novel insight: the administration
of MPL, compared with purified sphingomyelin, significantly increased the recovery of molecular species of ceramide containing d16:1 sphingosine in lymph. One possible
explanation for the improved bioavailability conferred by
MPL may be associated with their solubility in an aqueous
environment. MPL were comprised of 18.5 % sphingomyelin and 19.3 % phosphatidylcholine. Bile salts and phosphatidylcholine combine physiologically to form mixed
micelles which aid the solubility and absorption of dietary
fats and some drug molecules [19, 20]. Previous reports
revealed that curcuminoids co-formulated with phosphatidylcholine had improved bioavailability compared with
curcuminoids alone when administered to both humans and
rats [14, 15]. Also, the administration of phosphatidylcholine has been shown to enhance lymphatic absorption of
lycopene in rats [21]. It is therefore a possibility that physical properties, such as the higher solubility of the MPL
emulsion, inhibit sphingomyelin crystallization, resulting
in the increased bioavailability of dietary sphingomyelin.
Compared with sphingomyelin, the ingestion of MPL
not only increased maximum concentration of ceramides in
lymph, but decreased disappearance rate of ceramides from
lymph (Figs. 5, 6), even though changes of triacylglycerol and phospholipids levels in lymph were not different
between the sphingomyelin and the MPL group (Fig. 2).
These results indicated that the absorption of dietary sphingomyelin was regulated by a different pathway from that of
triacylglycerol and other phospholipids. Since it is not clear
how the MPL emulsion control the rate of sphingomyelin
absorption, further studies are needed to clarify the underlying mechanism.
The results of this study showed that the recovery rates
of ceramide moieties from purified sphingomyelin and
MPL ranged from 0.92 to 5.46 % and 0.68 to 4.20 %,
respectively. Previous reports demonstrated that the cumulative recovery of 4,8-sphingadienine from maize glucosylceramide was approximately 0.18 %, which is lower
than the recovery of the milk sphingomyelin in this study
[12]. In higher plants, the sphingoid base structure of
sphingolipids are more complicated than they are in mammals, and they contain 8-sphingenine, 4,8-sphingadienine,
4-hydroxy-8-sphingenine, etc. [22–24]. Sphingoid bases
originating from various dietary sources were substrates
for P-glycoprotein, which is a membrane bound transporter that mediates cellular efflux, or active transport, of
a wide range of structurally unrelated drugs and xenobiotics [25]. According to Sugawara et al. [26], the uptake of
sphingosine is significantly higher than the uptake of other
sphingoid bases, including 4,8-sphingadienine in differentiated Caco-2 cells, and P-glycoprotein probably contributes
to this selective absorption of sphingoid base. In addition,
after treatment with a P-glycoprotein inhibitor (verapamil),
995
4,8-sphingadienine accumulated in Caco-2 cells, but the
accumulation of sphingosine (d18:1) was not affected.
Thus, selective efflux of sphingoid bases by P-glycoproteins may be related to the difference in lymphatic absorption of dietary sphingolipids.
In conclusion, our results showed that dietary milk
sphingomyelin was incorporated into lymph as ceramide
structures and their molecular species were similar to
those hydrolyzed from milk sphingomyelin. Furthermore,
co-ingestion of dietary sphingomyelin with other glycerophospholipids, compared with purified sphingomyelin,
enhanced lymphatic absorption of milk sphingomyelin.
Our findings may be helpful in overcoming the very low
bioavailability of dietary sphingomyelin as well as promoting healthy effects.
Compliance with Ethical Standards Conflict of interest The authors declare that there are no conflicts
of interest.
References
1. Vesper H, Schmelz EM, Nikolova-Karakashian MN, Dillehay
DL, Lynch DV, Merrill AH Jr (1999) Sphingolipids in food and
the emerging importance of sphingolipids to nutrition. J Nutr
129:1239–1250
2. Zeisel SH, Char D, Sheard NF (1986) Choline, phosphatidylcholine and sphingomyelin in human and bovine milk and infant
formulas. J Nutr 116:50–58
3. Zeisel SH, Mar MH, Howe JC, Holden JM (2003) Concentrations of choline-containing compounds and betaine in common
foods. J Nutr 133:1302–1307
4. Watanabe S, Takahashi T, Tanaka L, Haruta Y, Shiota M,
Hosokawa M, Miyashita K (2011) The effect of milk polar
lipids separated from butter serum on the lipid levels in the liver
and the plasma of obese-model mouse (KK-Ay). J Funct Foods
3:313–320
5. Schmelz EM, Dillehay DL, Webb SK, Reiter A, Adams J, Merrill AH Jr (1996) Sphingomyelin consumption suppresses aberrant colonic crypt foci and increases the proportion of adenomas
versus adenocarcinomas in CF1 mice treated with 1,2-dimethylhydrazine: implications for dietary sphingolipids and colon carcinogenesis. Cancer Res 56:4936–4941
6. Schmelz EM, Bushnev AS, Dillehay DL, Liotta DC, Merrill AH
Jr (1997) Suppression of aberrant colonic crypt foci by synthetic
sphingomyelins with saturated or unsaturated sphingoid base
backbones. Nutr Cancer 28:81–85
7. Haruta Y, Kato K, Yoshioka T (2008) Dietary phospholipid concentrate from bovine milk improves epidermal function in hairless mice. Biosci Biotechnol Biochem 72:2151–2157
8. Haruta-Ono Y, Setoguchi S, Ueno HM, Higurashi S, Ueda N,
Kato K, Saito T, Matsunaga K, Takata J (2012) Orally administered sphingomyelin in bovine milk is incorporated into skin
sphingolipids and is involved in the water-holding capacity of
hairless mice. J Dermatol Sci 68:56–62
9. Morifuji M, Oba C, Ichikawa S, Ito K, Kawahata K, Asami Y,
Ikegami S, Itoh H, Sugawara T (2015) A novel mechanism for
improvement of dry skin by dietary milk phospholipids: effect
13
996
on epidermal covalently bound ceramides and skin inflammation
in hairless mice. J Dermatol Sci 78:224–231
10. Nilsson A (1968) Metabolism of sphingomyelin in the intestinal
tract of the rat. Biochim Biophys Acta 164:575–584
11. Schmelz EM, Crall KJ, Larocque R, Dillehay DL, Merrill AH Jr
(1994) Uptake and metabolism of sphingolipids in isolated intestinal loops of mice. J Nutr 124:702–712
12. Sugawara T, Tsuduki T, Yano S, Hirose M, Duan J, Aida K,
Ikeda I, Hirata T (2010) Intestinal absorption of dietary maize
glucosylceramide in lymphatic duct cannulated rats. J Lipid Res
51:1761–1769
13. Byrdwell WC, Perry RH (2007) Liquid chromatography with
dual parallel mass spectrometry and 31P nuclear magnetic resonance spectroscopy for analysis of sphingomyelin and dihydrosphingomyelin. II. Bovine milk sphingolipids. J Chromatogr
A 1146:164–185
14. Cuomo J, Appendino G, Dern AS, Schneider E, McKinnon TP,
Brown MJ, Togni S, Dixon BM (2011) Comparative absorption
of a standardized curcuminoid mixture and its lecithin formulation. J Nat Prod 74:664–669
15. Jager R, Lowery RP, Calvanese AV, Joy JM, Purpura M, Wilson
JM (2014) Comparative absorption of curcumin formulations.
Nutr J 13:11
16. Blaas N, Schuurmann C, Bartke N, Stahl B, Humpf HU (2011)
Structural profiling and quantification of sphingomyelin in
human breast milk by HPLC–MS/MS. J Agric Food Chem
59:6018–6024
17. Green PH, Glickman RM (1981) Intestinal lipoprotein metabolism. J Lipid Res 22:1153–1173
13
Lipids (2015) 50:987–996
18. Green PH, Riley JW (1981) Lipid absorption and intestinal lipoprotein formation. Aust N Z J Med 11:84–90
19. Carey MC, Small DM (1978) The physical chemistry of cholesterol solubility in bile. Relationship to gallstone formation and
dissolution in man. J Clin Invest 61:998–1026
20. Hegardt FG, Dam H (1971) The solubility of cholesterol in
aqueous solutions of bile salts and lecithin. Z Ernahrungswiss
10:223–233
21. Nishimukai M, Hara H (2004) Enteral administration of soybean phosphatidylcholine enhances the lymphatic absorption
of lycopene, but reduces that of alpha-tocopherol in rats. J Nutr
134:1862–1866
22. Sperling P, Zahringer U, Heinz E (1998) A sphingolipid desaturase from higher plants. Identification of a new cytochrome b5
fusion protein. J Biol Chem 273:28590–28596
23. Sperling P, Libisch B, Zahringer U, Napier JA, Heinz E (2001)
Functional identification of a delta8-sphingolipid desaturase
from Borago officinalis. Arch Biochem Biophys 388:293–298
24. Sugawara T, Duan J, Aida K, Tsuduki T, Hirata T (2010) Identification of glucosylceramides containing sphingatrienine in maize
and rice using ion trap mass spectrometry. Lipids 45:451–455
25. Ueda K, Yoshida A, Amachi T (1999) Recent progress in P-glycoprotein research. Anticancer Drug Des 14:115–121
26. Sugawara T, Kinoshita M, Ohnishi M, Tsuzuki T, Miyazawa T,
Nagata J, Hirata T, Saito M (2004) Efflux of sphingoid bases by
P-glycoprotein in human intestinal Caco-2 cells. Biosci Biotechnol Biochem 68:2541–2546