ORIGINAL ARTICLE
Analysis of fatty acid composition in human bone marrowaspirates
Ryota Deshimaru, Ken Ishitani, Kazuya Makita, Fumi Horiguchi and Shiro Nozawa
Department of Obstetrics and Gynecology, School of Medicine, Keio University, Tokyo, Japan(Received for publication on January 17, 2005)(Revised for publication on June 6, 2005)(Accepted for publication on June 16, 2005)

Abstract. In the present study, the fatty acid composition of bone marrow aspirates and serum phospholipids in nine patients with hematologic diseases was investigated, and the effect of fatty acids on
osteoblast differentiation in ST2 cells was examined. The concentrations of oleic acid and palmitic acid
were signiﬁcantly higher in bone marrow aspirates than in serum phospholipids, but the concentrationsof other fatty acids did not differ. The rate of alkaline phosphatase positive ST2 cells induced by BMP2 was signiﬁcantly increased by oleic acid, but was unaffected by the presence or absence of palmitic
acid. We conclude that the fatty acid composition of bone marrow aspirates differs from that of serum
phospholipids. This difference may affect osteoblast differentiation in the bone marrow microenvironment. (Keio J Med 54 (3): 150–155, September 2005)
Key words: fatty acids, osteoporosis, osteoblast, bone marrow

Introduction
Osteoporosis is a widespread public health problem
among postmenopausal women because of the association between low bone mass and the occurrence ofvertebral and hip fractures.1 Bone mass is maintained
locally by a balance between bone formation by osteoblasts and bone resorption by osteoclasts. Osteoblasts
derived from mesenchymal tissues replace resorbedbone by elaborating matrix, which then becomes mineralized.2 After menopause, bone resorption exceedsbone formation. Consequent bone loss accompanied byan increased volume of adipose cells in bone marrow
may inhibit osteoblast proliferation.3 Numerous factors, including bone morphogenetic protein-2 (BMP2),4 transforming growth factor beta (TGF-b),5 andretinoic acid,6 regulate osteoblastic bone formation.
The secretion of many of these factors, including fatty
acids, is affected during menopause.
Previous studies have documented that dietary fat
plays an important role in skeletal biology and bonehealth.7 Fatty acids may inﬂuence bone metabolism byaltering the biosynthesis of prostaglandin (PGE2 ).8–10
In particular, n-3 polyunsaturated fatty acid (PUFA)

increases bone formation by decreasing PGE2 biosynthesis.11 Thus, numerous studies have reported theeffects of dietary fat on bone formation in vitro or invivo.7,12,13 However, little is known about the association between fatty acid composition and the microenvironment of human bone marrow, which may inﬂuence osteoblastic bone formation. Moreover, the effectsof fatty acids in bone marrow on osteoblast differentiation are not fully understood.
In order to elucidate the mechanism of postmenopausal osteoporosis, we analyzed the fatty acid composition of bone marrow aspirates and serum phospholipids in nine patients with hematologic diseases and thenexamined the effects of fatty acids on osteoblast differentiation, using a mouse mesenchymal cell-derived ST2cell line.
Methods
Subjects
Nine female patients with hematologic diseases who
were referred to the Department of Internal Medicineof Saitama Medical School Hospital between 1999 andReprints requests to: Dr. Kazuya Makita, Department of Obstetrics and Gynecology, School of Medicine, Keio University, 35 Shinanomachi,
Shinjuku-ku, Tokyo 160-8582, Japan150

Keio J Med 2005; 54 (3): 150–155

2001, participated in this study. The patients who havereceived bone marrow transplants or had myelosupressive chemotherapy within three months were precluded from theses subjects. Oral and written informed
consent was obtained from each subject before study
entry. The study was conducted in accordance with ourinstitutional guidelines. To analyze fatty acid composition, we examined a portion of the serum and the bonemarrow aspirates obtained from the patients.
Fatty acid analysis
The fatty acid composition of the bone marrowaspirates and serum phospholipids were analyzed as
described previously.14–16 The phospholipid fraction
was isolated from the extracted lipids with the use of aone-dimensional neutral lipid solvent system of hexane,
diethyl ether, and acetic acid (87 : 2 : 1, v/v). The phospholipid fraction was scraped into glass tubes, andphospholipid fatty acid methyl esters were preparedby transmethylation with 2 M KOH-ethanol and 6
M HCL-ethanol. Fatty acid methyl ester derivatives
formed from the isolated phospholipid fraction wereseparated by gas-liquid chromatography (HewlettPackard 5890). Individual fatty acid methyl esters wereidentiﬁed by comparing sample peak retention times
with n-pentadecane (Tokyo Kasei Kogyo). The results
were expressed as the relative percentage of total identiﬁed fatty acids.
Cell culture and alkaline phosphatase assays
Mouse mesenchymal cell-derived ST2 cell line waspurchased from RIKEN Cell Bank. The cells weremaintained at subconﬂuence in Dulbecco’s modiﬁed
Eagle’s medium (DMEM) with 10% fetal bovine serum(GIBCO BRL). To evaluate adipocyte differentiation,
cells were grown to conﬂuence in DMEM with 10%
FBS. The culture medium was supplemented with 10 7
M rosiglitazone (Calbiochem) to allow adipocyte differentiation. Cells were seeded at a density of 4,000
cells per well in 96-well dishes and allowed to grow to
conﬂuence for 24 hours. After 7 days of treatment withrosiglitazone, lipid staining was done using oil-red O
soluble dye, which stains neutral lipids. To examine
osteoblast differentiation, cells were grown to conﬂuence in DMEM with 10% FBS. The culture medium
was supplemented with BMP-2 to allow osteoblast
differentiation. In the same way as BMP-2, retinoic
acid, TGF-b, and fatty acids were added to the culture
medium for 7 days. Recombinant human BMP-2 wasprovided by Yamanouchi Pharmaceutical Co., Ltd.
Alkaline phosphatase assays were performed using a
Sigma–Aldrich diagnostic kit. Cells were seeded at adensity of 2:0  10 4 cells per well in 24-well dishes and151

allowed to grow to conﬂuence for 24 hours. After 7
days of treatment with rh BMP-2, the cells were lysedwith 200 ml of 0.01% SDS in PBS, and the lysates were
scraped and collected. Fifty microliters of lysate wasadded to 50 ml of prewarmed p-nitrophenol disodium
phosphate (PNPP) substrate with alkaline phosphatase(ALP) buffer solution (SIGMA 221), and the samples
were incubated at 37 C. The reaction was stopped byadding 50 ml of 2 N NaOH, and the optical density(OD) was read at 405 nm. The ALP activity was determined by comparison with a PNPP standard curve. All
values were normalized against the protein concentration determined with the use of TAKARA BCA Protein Assay Reagent.
Statistical analysis
The data are expressed as means G standard deviation (SD). Statistical analysis was done by the two-tailed
Student’s t-test for two unpaired groups. Differenceswith p values of less than 0.05 were considered statistically signiﬁcant.
Results
Fatty acid composition of the human bone aspirates andserum phospholipids
Table 1 shows the clinical characteristics of thepatients who underwent bone marrow puncture. All
nine female patients had hematologic diseases and despite having different diseases, the fatty acid composition did not differ substantially among these patients.
The concentrations of the fatty acids of the bone aspirates and serum phospholipids are shown in Table 2.
The concentrations of oleic acid and palmitic acid weresigniﬁcantly higher in bone marrow aspirates than inTable 1

Clinical Characteristics of the Subjects

Patientno.

Age(yr)/sex

Pre/post
menopause

DiseaseTC(mg/dl)LDL-C(mg/dl)

1
2
3
4
5
6
7
8
9

33/female
81/female
44/female
61/female
49/female
68/female
65/female
40/female
71/femalepremenopause
postmenopausepremenopause
postmenopause
postmenopause
postmenopause
postmenopausepremenopause
postmenopause

AML
CML
CLL
NHL
NHL
NHL
MDS
myeloma
NHL

200.1
243.3
165.0
188.8
156.3
282.4
238.5
170.1
252.2

130.4
146.4
112.3
128.0
115.0
186.5
152.4
111.9
159.7

AML: acute myeloid leukemia, CML: chronic myelogenous leukemia,
CLL: chronic lymphocytic leukemia, NHL: non-Hodgkin’s lymphoma,
MDS: myelodysplastic syndromes, TC: total cholesterol, LDL-C: lowdensity lipoprotein cholesterol.

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Deshimaru R, et al: Fatty acid composition in human bone marrow

Table 2 Fatty Acid Composition of Bone Marrow Aspirates andSerum Phospholipids
Fatty acids

Bone marrowaspirates(10 2 mEq/l)Serumphopholipids(10 2 mEq/l)ppalmitic acidstearic acidoleic acidlinoleic acidlinolenic acidarachidic acideicosenoic acideicosapentaenoic acidbehenic aciddocosapentaenoic aciddocosahexaenoic acidlignoceric acidnervonic acidtotal

20:75 G 3:79
6:65 G 1:53
16:99 G 4:03
11:29 G 3:89
0:72 G 0:38
0:10 G 0:04
0:37 G 0:36
0:56 G 0:52
0:19 G 0:05
0:47 G 0:17
2:50 G 1:23
0:10 G 0:03
0:12 G 0:10
66:78 G 18:53

16:90 G 3:50
7:82 G 2:03
12:02 G 3:04
13:82 G 4:01
0:61 G 0:26
0:14 G 0:05
0:34 G 0:32
0:68 G 0:42
0:23 G 0:11
0:39 G 0:30
3:32 G 1:87
0:16 G 0:13
0:20 G 0:17
64:14 G 15:02

p < 0.05
NS
p < 0.05
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS

Values expressed as means G SD (standard deviation). Fatty
acids < 0.10 (10 2 mEq/l) are not shown.

serum phospholipids. There were no marked differences in the concentrations of other fatty acids betweenbone marrow aspirates and serum phospholipids. These
data indicated that the concentrations of oleic acid andpalmitic acid primarily differed between bone marrowaspirates and serum phospholipids.
Osteoblast and adipocyte differentiation potential of ST2
cells

ALP assay and oil-red O staining of mesenchymal ST2
cells were performed with or without TGF-b and retinoic acid, both of which were known to allow osteoblast
differentiation,17 in order to verify the osteoblast andadipocyte differentiation potentials of ST2 cells which
we used. Fig. 1 shows the morphology of ST2 cells in
the presence or absence of 10 6 M rosiglitazone, andtreatment of ST2 cells with rosiglitazone induced adipogenic differentiation with accumulation of lipid. The
rates of ALP positive cells were signiﬁcantly increased
in a dose-dependent fashion by TGF-b and retinoic acid
(Fig. 2). In contrast, the rates of oil-red O positive cells
were signiﬁcantly decreased in a dose-dependent fashion by retinoic acid, but were unchanged by treatment
with TGF-b (Fig. 3).
Effects of oleic acid and palmitic acid on osteoblast differentiation of ST2 cells
To test the effects of oleic acid and palmitic acidon osteoblast differentiation, we performed ALP assays
using ST2 cells induced by BMP-2. The rate of ALP
positive cells was signiﬁcantly increased by 100 mM ofoleic acid plus 100 ng/ml of BMP-2. The rate of ALP
positive cells was similar with or without 100 mM ofpalmitic acid (Fig. 4), and the rate of oil-red O positive
cells was also similar in all of these conditions (Fig. 5).
These ﬁndings indicated that only oleic acid acted synergistically with BMP-2 to promote osteoblastic differentiation of ST2 cells.
Discussion

Then, we selected oleic acid and palmitic acid, whichshowed signiﬁcantly higher concentrations in bone marrow aspirates than in serum phospholipids (Table 2).

Fig. 1

Mesenchymal stem cells in bone marrow, whose
progeny become committed to speciﬁc lineages in re-

Oil-red O stainig of lipids in ST2 cells in the absence (A), and in the presence (B) of 10 6 M rosiglitazone. Arrows indicate lipids.

Keio J Med 2005; 54 (3): 150–155

153

Fig. 2 Osteoblast differentiation potential of ST2 cells with dosedependent induction by TGF-b and retinoic acid. *p < 0:05: Signiﬁcantly different from basal value.
Fig. 4 Effects of oleic acid and palmitic acid on osteoblast differentiation of ST2 cells with induction by 100 ng/ml BMP-2. *p < 0:05
(BMP-2 vs BMP-2 plus oleic acid): Signiﬁcant differences from theculture incubated with 100 mM of oleic acid are indicated.

Fig. 3 Effects of TGF-b and retinoic acid on adipocyte differentiationof ST2 cells with induction by 10 7 M rosiglitazone. *p < 0:05: Signiﬁcantly different from basal value.

sponse to growth factors, hormones, and other signals,
are pluripotential and differentiate into ﬁbroblasts, adipocytes, chondrocytes, or osteoblast.18–23 ST2 cell line,
a clone of stromal cells, was isolated from the bonemarrow of BC8 mice. This cell line has characteristics
typical of preadipocytes and differentiates into osteoblasts and adipocyte-like mesenchymal stromal cells.23
ST2 cells differentiate into osteoblasts when BMP-2,
TGF-b, and ascorbic acid are added to the culture medium. BMP-2 belongs to the TGF-b superfamily, which
induces the development and differentiation of bonecells. ST2 cells also differentiate into adipocytes whenlong-chain fatty acids are added to the culture medium.25,26 Peroxisome proliferator-activated receptor gsimultaneously promotes the formation of adipocytes
and suppresses the formation of osteoblasts,18 and retinoic acid also simultaneously promotes the formation of osteoblasts and suppresses the formation of
adipocytes.6
In this study, using ST2 cells, the effects of TGF-b or
retinoic acid on osteoblast and adipocyte differentiation
were consistent with previous ﬁndings.6 We analyzed

Fig. 5 Effects of oleic acid and palmitic acid on adipocyte differentiation of ST2 cells with induction by 10 7 M rosiglitazone.

the fatty acid composition of the culture medium usedin this study, which contained the same lot of 10% FBS(data not shown). The medium was found to containminimal amounts of oleic acid and palmitic acid. It wastherefore considered to have had no appreciable effecton the results of our differentiation experiments. Our
assay using ST2 cells was therefore considered a valid
approach for examining the effects of fatty acids on
osteoblast or adipocyte differentiation.
We had initially thought that oleic acid and palmitic
acid have simultaneous differentiation effects to promote adipocyte differentiation and suppress osteoblast
differentiation. Several reasons supported this assumption: 1) Mature adipocytes release PUFA, which impairs
osteoblast proliferation,3 and long-chain fatty acids,
which promote adipocyte differentiation. 2) The bone

154

marrow microenvironment is a complex network ofcells and extracellular matrix that maintains the hematopoietic system throughout the life of the individual.
Regulating factors may maintain the homeostasis ofmesenchymal cell differentiation in the bone marrow
microenvironment. 3) Our results indicated that the ratios of oleic acid and palmitic acid were higher in bonemarrow than in serum phospholipids.
Contrary to our expectation, our result showed that
oleic acid acts synergistically with BMP-2 in promoting
osteoblast differentiation of ST2 cells. In addition, theresults of oil–red O staining of ST2 cells indicatedno signiﬁcant promotion or suppression of adipocyte
differentiation by oleic acid. These results indicated that
oleic acid may promote osteoblast differentiation of
only pre-osteoblasts synergistically with BMP-2, because BMP-2 has osteoblast differentiation effect on
both mesenchymal cells and pre-osteoblasts with theinduction of Runx2 expression.27 Oleic acid, a monounsaturated fatty acid, is found abundantly in palm oil
and olive oil. Mono- or polyunsaturated fatty acids havebeen shown to have protective effects towards the development of cardiovascular disease; however, the relation between monounsaturated fatty acids and osteoporosis is still unknown.7 From a clinical standpoint, inorder to propose ideal eating habits and dietary recommendations to prevent osteoporosis, it is necessary
to elucidate the role of oleic acid in bone metabolism.
To our knowledge, this is the ﬁrst study to show
differences in fatty acid composition between bonemarrow aspirates and serum phospholipids. We studied
subjects who had never received bone marrow transplants, which impair differentiation of bone marrowstromal cells into osteoblasts,28 or had myelosupressive
chemotherapy within three months.
Our study had several limitations. First, it was asmall study, with differences in age and menopausal
status of the subjects. Second, we did not measure bonemineral density in our patients. Third, there could beconsiderable effects on the bone marrow function of
our subjects who had hematologic diseases and received
myelosupressive chemotherapy previously, and thecharacteristics of our subjects do not reﬂect those of
women in the general population. Indeed, the ideal
subjects would have been healthy individuals, but bonemarrow puncture is a painful procedure considered
ethically unacceptable in a study of healthy volunteers.
Despite these limitations, our results suggest a difference in fatty acid composition between bone marrowaspirates and serum phospholipids. Further studies, if
possible using samples of bone marrow aspirates obtained from a bone marrow bank, are required to verify
our results. In conclusion, our ﬁndings suggest that the
fatty acid composition of bone marrow aspirates differsfrom that of serum phospholipids. This difference pre-Deshimaru R, et al: Fatty acid composition in human bone marrow

sumably affects osteoblast differentiation in the bonemarrow microenvironment.
Acknowledgments: The authors would like to thank Mitsubishi
Kagaku Bio-Clinical Laboratories for analyzing the fatty acid composition of the bone marrow aspirates.

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