Journal of Chromatography B 1152 (2020) 122259

Contents lists available at ScienceDirect

Journal of Chromatography B

journal homepage: www.elsevier.com/locate/jchromb

Optimization and validation of a chiral CE-LIF method for quantitation of

aspartate, glutamate and serine in murine osteocytic and osteoblastic cells
Maria Paz Lorenzoa, Luis Valientea, Irene Buendiab, Arancha R. Gortázar b, Antonia Garciaa,⁎
a
Center for Metabolomics and Bioanalysis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities. Campus Montepríncipe, Boadilla del Monte,
28668 Madrid, Spain
b
Bone Physiopathology Laboratory, Applied Molecular Medicine Institute (IMMA), Facultad de Medicina, Universidad San Pablo-CEU, CEU Universities, Campus
Montepríncipe, Boadilla del Monte, 28668 Madrid, Spain

ARTICLEINFO ABSTRACT
Keywords: Asp, Glu, and D-Ser are chiral amino acids and neurotransmitters binding to the N-methyl-D-aspartate receptor (NMDA) and
Chiral analysis they participate in glutamate signalization. D-amino acids are increasingly being recognized as important signaling molecules
Capillary electrophoresis and variations in their levels are considered a marker of different pathologies, however, there is still a lack of knowledge
Fluorescence detection about the role of most of D-amino acids in living organisms such as bone cells. A method for determination of
Osteocytes concentrations of L/D-Asp, L/D-Glu and L/D-Ser in two types of bone cell lines: murine osteocytes (MLOY4) and osteoblasts
Osteoblasts
(MC3T3-E1) is presented. It is based on capillary electrophoresis coupled to laser-induced fluorescence detection in normal
Neurotransmitters
polarity with 4-fluoro-7-nitro-2,1,3-benzoxadiazole as derivatizing agent suitable for an Argon ion laser source. The
electrolyte consists of 137.5 mM borate buffer and 12.5 mM β-cyclodextrins as chiral selectors and the separation lasts 25
min. The method was optimized and validated for specificity, sensitivity, linearity, accuracy, and precision in murine
osteocytes and osteoblasts. LLOQ was 0.25 µmol L −1 for the three D-amino acids and linearity was confirmed with r > 0.995
for all D-and L-amino acids. Accuracy ranged between 81.9% and 111.7% and intra-day precision ranged between 1.8% and
10.9%. Concentrations of D- and L- Asp, Glu, and Ser are given and statistical differences between osteocytes and
osteoblasts were found. The highest differences corresponded to L- and D-Glu. This method could play a fundamental role in
the study of therapeutic targets in the treatment of bone diseases.

1. Introduction L-Glu is the main excitatory neurotransmitter of the central

Amino acids AAs are metabolites and the base of signaling
molecules, hormones, enzymes, antibodies, receptors and
numerous protein architectures in living organisms. Alpha-amino
acids, α-AAs, except Glycine are chiral compounds being L-AAs the
most abundant in tissues. D-AA isomers are increasingly being
nervous system and it is involved in the metabolism of lipids and
sugars, as well as in potassium transport through the blood-brain
barrier, and in wound regeneration. In addition to the NMDA
receptor, it binds to several ionotropic (iGluR) and metabotropic
(mGluR) receptors [5]. D-Glu has been found in the brain of
mammals, such as rats, although in very low concentrations [6,7].

⁎
Corresponding author at: Center for Metabolomics and Bioanalysis (CEMBIO), Facultad de Farmacia, Universidad CEU San Pablo, CEU Universities, Campus Monteprincipe, Boadilla
del Monte, 28668 Madrid, Spain. E-mail address: antogar@ceu.es (A. Garcia).

https://doi.org/10.1016/j.jchromb.2020.122259
Received 1 March 2020; Received in revised form 20 June 2020; Accepted 29 June 2020
Available online 03 July 2020
1570-0232/ © 2020 Elsevier B.V. All rights reserved.
recognized as important signaling molecules and variations in their
levels are considered a marker of different pathologies, however,
there is still a lack of knowledge about the role of most of them in
the organisms [1].
Aspartic acid Asp, glutamic acid Glu, and D-serine D-Ser are
proteinogenic and non-essential AAs and they are three of the
main neurotransmitter AAs, binding to the N-methyl-D-aspartate
receptor (NMDA) and taking part in glutamate signalization [2]. L-
Asp participates in antibody production, hepatic function, and
energetic metabolism. D-Asp is a neurotransmitter and it has been
associated with neurogenesis, reproduction and visual function [3].
Both enantiomers are closely related to L- and D-Glu, being
metabolized by the same enzyme, the D-aspartic acid oxidase [4].
D- Ser is the best-known D-amino acid, it is synthesized from its L-
form through serine racemase. It is an endogenous agonist of the
NMDA receptor, and its disturbances have been associated with
schizophrenia and depression, while its administration has shown
antidepressant effects in clinical assays with rats [3,8–10].
Osteocytes are long-lived cells that play an essential role in
mature bone. They are the main bone receptors of mechanical
signals and
regulate both bone formation and resorption through the
production of Sclerostin – a key inhibitor of Wnt signaling in
osteoblasts, and of the Receptor Activator of Nuclear factor
Kappa-Β Ligand (RANKL), responsible of osteoclast differentiation
and activation [11].
M.P. Lorenzo, et al.
Through the NMDA receptor, L-Glu regulates the homeostasis
of calcium and phosphate and mediates mechanical signalization
in bone [11,12]. Moreover, L and D-Ser regulate osteoclast
differentiation and osteocytes maturation respectively [8,12–14].
Because of their longevity, osteocytes are more likely than
osteoclasts or osteoblasts to accumulate molecular damage over
time.
Similarly, the role of osteoblasts is prevalent in bone
formation, and matrix production and mineralization. They are
essential in the prevention of age-related bone loss, which
appears when their function is affected and they are incapable to
counterbalance the bone resorption by osteoclasts [11,12,14,15].
Some studies have unveiled the importance of certain
neurotransmitters in this process, through the previously
mentioned NMDA receptor. L-Glu has been shown to activate
osteoblast differentiation and proliferation and to reduce
osteoclast adhesion [5,15,16]. Therefore, the usage of glutamate
agonists and antagonists has been suggested in the context of
musculoskeletal diseases [16,17]. Under that scenario, the role of
their counterparts D-Glu and D-Asp is still unknown, likely due to
the very small range of concentrations of D-AAs in cells and
tissues (nM).
The analysis of trace amounts of D-AAs in biological samples
requires the use of very sensitive approaches such as HPLC, GC
and capillary electrophoresis, CE, coupled to fluorescence or
mass spectrometry detectors [4,17–20]. Amino acids are very
polar compounds and the lack of chromophore hinders their
reliable analysis and that is extremely important for D-AAs. This
may be solved by adding a derivatizing agent to produce a
fluorescent derivate and less polar and bigger molecule instead
of the direct strategy which is based on the use of chiral selectors
or chiral stationary phases for the separation of native AAs.
Regarding the addition of derivatizing agents, they could be a
chiral labeling agents CDA, such as 6-aminoquinolyl-N-
hydroxysuccinimidyl carbamate (AQC) [21] so the separation coul
be based on non-chiral mechanisms. When non-chiral reagents
are selected, such as dansyl chloride (DNS-Cl) [22], 4-fluoro-7-
nitro-2,1,3-benzoxadiazole (NBD-F), 9-
fluorenylmethyloxycarbonyl chloride (FMOC-Cl), fluorescein
isothiocyanate (FITC), 2,3-naphthalenedicarboxaldehyde (NDA)
[23], or o-phthalaldehyde (OPA) [24] then chiral separation
mechanisms are required. Long incubation time or by-products
that interfere with the analysis are some of the main drawbacks
of reagents such as FITC or AQC [25].
CE is especially suitable for the analysis of polar compounds
in aqueous matrices, such as biological samples, and it displays
several advantages such as low operation cost and
environmentally friendly, making its use very practical in
developing countries. Besides, low sample volume requirements
and short analysis time are also remarkable. It can also be
coupled with different detection systems, such as laser-induced
fluorescence, LIF, while also being compatible with chiral
selectors for chiral separation of enantiomers of the same
compound. The most used chiral selectors include streptomycin,
oligonucleotides, maltodextrins, chiral interleukins or native or
modified cyclodextrins CD (α, β or γ). By far, β-CD are the most
common ones [26], and as they were previously selected by our
research group for the analysis of AAs, they were chosen as chiral
selectors now for chiral analysis of L and D- AAs.
NBD-F is a derivatizing agent with several advantages: It
requires short incubation time with moderate temperature and
alkaline pH to react with primary and secondary amines,
generating a single derivate for each enantiomer [27–29].
Moreover, it is compatible with an argon- ion laser source, λex:
488 nm, λem: 522 nm). Besides, our group has a successful
Journal of Chromatography B 1152 (2020) 122259
experience with it, so it was selected as a derivatizing agent for
the current research.
Here we have optimized and validated a method for the chiral
analysis of three L ɑ-AAs neurotransmitters in murine bone cell
lines in less than 25 min and then it was applied to real samples.
The method is based on the use of 4-fluoro-7-nitro-2,1,3-
benzoxadiazole NBD-F as a labeling agent combined with CE-LIF
and β-CD as chiral selectors. To our knowledge, it is the first time
that concentrations of D-Asp, D-Glu, and D-Ser are obtained in cell
lines of osteocytes and osteoblasts.
2. Materials and methods
2.1. Reagents
D-Asp, L-Asp, D-Glu, L-Glu, D-Ser, L-Ser, α-aminoadipic acid and
β- CD with purity > 99.0% were purchased from Sigma (Steinheim,
Germany) and NaOH analytical grade from Panreac Química S.A.U.
(Barcelona, Spain). Boric acid 99.5% and methanol (MeOH) 99%
HPLC- grade were purchased from Sigma–Aldrich (Steinheim,
Germany) and HCl 35.5% (w/w) from Fluka (Buchs, Switzerland). 4-
fluoro-7-nitro- 2,1,3-benzoxadiazole (NBD-F) 99% from TCI (Tokio,
Japan). Ultra-pure water was obtained using a Milli-Q plus system
from a Milli-Qplus185 (Millipore, Billerica MA). All other materials
and reagents were of analytical grade.
2.2. Standard solutions preparation
Independent 25 mmol L−1 stock solutions were prepared for
each D and L α-AA with ultra-pure water, and from them,
respective 1 mmol L−1 intermediate solutions were prepared by
dilution. All solutions were stored at −20 °C until analysis. 2.3. Cell
culture and sample preparation
The osteocytic MLO-Y4 cell line, derived from murine long
bones (kindly supplied by Dr. Lynda Bonewald, University of
Indiana, Indianapolis, IN) were grown in Minimum Essential
Medium with alpha modifications, α-MEM, supplemented with
2.5% fetal bovine serum (FBS), 2.5% calf serum (CS) and 1%
penicillin–streptomycin in 5% CO2 at 37 °C. Mouse osteoblastic
MC3T3-E1 cells (CRL-2593; American Type Culture Collection,
Mannasas, VA) were grown in α-MEM supplemented with 10%
FBS, 1% penicillin–streptomycin and 2 mM glutamine in 5% CO 2 at
37 °C. Both cell lines were grown in Petri dishes of 100 × 20 mm
[30]. 500 µL of lysis buffer RIPA (Sigma-Aldrich, St. Louis, MO, USA)
was added to obtain cellular lysates with 2x106 cells of each line.
Samples were stored at −80 °C until the day of analysis.
2.4. Derivatization process
Samples were prepared according to our previous papers
[19,31] with some modifications for this type of samples. In brief,
in a glass tube, 40 µL of the cellular lysate or the working standard
solution was mixed with 20 µL of 100 µmol.L −1 α-aminoadipic acid
as internal standard (IS), 50 µL of 40 mM NBD-F solution
(methanol: 500 µmol L−1 HCl, 1:1 v/v), and 150 µL of 10 mM
aqueous borate buffer pH 10.0. The mixture was vortexed for 1
min and incubated at 60 °C for 15 min.
2.5. CE-LIF method
A P/ACE System MDQ (Beckman Instruments, Fullerton, CA,
USA) coupled with a laser-induced fluorescence detector and an
Argon ion source λex = 488 nm, λem = 522 nm was used. The
separation was performed with a bare fused silica capillary
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M.P. Lorenzo, et al. Journal of Chromatography B 1152 (2020) 122259
(Beckman Coulter, Madrid, Spain), with 75 μm of ID and a total as described in section 2.3. Taking into account the sample matrix,
length of 60 cm. and the previous methods developed by our group [19,31] for
The method was based on our previous papers [19,31] but amino acid screening in urine and plasma samples, further method
after optimization for the chiral analysis. In brief, injections were optimization was performed with a BGE consisted of borate buffer
made at the anodic end with a pressure of 0.5 psi (33 mbar) for 15 at pH 10.25 and 12.5 mM β-CD. We confirmed that when the
s. Background electrolyte (BGE) consisted of 137.5 mM borate concentration of borate buffer was 90 mmol L −1 [19] the resolution
buffer at pH 10.25 (pH adjusted with 2 M NaOH) and 12.5 mM β- between the pairs D/L for Glu and Asp was very poor and the
CD. The applied voltage was +30 kV and the capillary temperature analysis time was 40 min. Besides, when the concentration of the
was 17 °C. The current observed under these conditions was +143 borate buffer was 175 mmol L−1 [31] the separation for D/L-Ser in
µA. plasma resulted improved but the analysis time was higher than 50
Between the runs, the capillary was flushed at 20 psi with 0.1 min for Asp and Glu (data not shown). Considering that, the
M HCl (3 min), water (2 min), and BGE (3 min). The buffer vials method was optimized for the concentration of the separation
were refreshed after every six analysis to maintain consistency. buffer to achieve the best resolution and within short analysis
time.
2.6. Validation study Table 1
Resolution values on critical separations of through the increase of running
The method was validated for selectivity, linearity, LLOQ and buffer concentration. Abbreviations: Ser: serine; Ala: alanine; Asp: aspartic
acid. tm: migration time; R: resolution; [c.o.]: current observed.
LOD, accuracy, instrumental precision and method precision (with
real samples). Specificity was studied by comparing the Buffer 125 mM Buffer 137.5 mM Buffer 150 mM
electropherograms obtained after NBD-F derivatization of i) RIPA
[c.o. = 136 µA] [c.o. = 142 µA] [c.o. = 162 µA]
lysis buffer without IS, ii) sample blank with IS, iii) the mixture of
Analyte tm (min) R(USP) tm (min) R(USP) tm (min) R(USP)
standards, iv) lysate from osteocytes and v) lysate from
osteoblasts. L-Ser 11.30 2.05 12.63 2.37 14.17 3.01

The linearity of the relative response for standards was studied D-Ser 11.62 13.06 14.70
by assaying in triplicate 5 levels of concentrations for each D-AA
L- Glu 15.15 2.66 17.23 3.42 20.21 4.81
and L-AA covering from 0.25 to 5.00 µmol L−1 and from 2.5 to 50.0 D-Glu 15.79 18.15 21.47
µmol L−1 respectively. The individual ranges are 0.25, 0.50, 1.00,
1.50 and 2.50 µmol L−1 and 2.5, 5.0, 10.0, 15.0 and 25.0 µmol L −1 for L-Asp 19.32 0.95 23.17 1.45 29.30 1.60
D- and L- AAs respectively. D- Asp 19.57 23.54 29.95
The limits of detection LOD were estimated using the standard
deviation from the y-intercept (Sa) of the calibration curve, using For the method optimization, a mixture with 50 µmol L −1 L-Glu
the following equation. Then, the lower limits of quantitation, and L-Ser, 12.5 µmol L−1 L-Asp, 25 µmol L−1 D-Glu and D-Ser, and
LLOQs, were tested experimentally for precision and accuracy. 12.5 µmol L−1 D-Asp were prepared. Higher borate concentration in
the BGE increases the analyte migration time but also the
3 separation between coeluting peaks. However, it also broadens
× Sa LOD
= the analyte plug and decreases the sensitivity. Thus, it is necessary
S to find the concentration that allows good resolution and
l sensitivity between L/D enantiomers with appropriated analysis
o time. Different borate buffer concentrations were assayed: 125,
p
137.5 and 150 mM, Table 1. While Glu and Asp contains an
e
additional carboxyl group, Ser has a hydroxyl; so the lesser
Accuracy was checked by comparing in triplicate the values negative charge of Ser at pH 10.25 decreases the electrophoretic
obtained in spiked samples prepared into the linearity range, mobility towards the anodic electrode, and thus it elutes the first
considering the endogenous concentrations previously measured one. Both L/D-Asp and L/D-Glu have a similar negative charge but
in the samples. 6 different concentrations were added 0.25, 0.50, D-Asp displays the highest charge/size ratio, so it will be the most
1.00, 1.50, 2.00 and 2.50 µmol L−1 for D-AAs and 2.5, 5.0, 10.0, 15.0 attracted by the anodic electrode, resulting in the longest
and 25.0 µmol L−1 for L-AAs. The matrix effect was evaluated by migration time.
comparing the slopes from the linearity curves obtained with As depicted in Table 1, borate concentration of 137.5 and 150
samples and standards with the following equation: mM produces better resolution between enantiomers than 125
Samples lin slope. mM, especially for the pair L/D-Asp. The 137.5 mM borate buffer
%Matrix effect = 100 × (1 ) was selected because it gives good separation between the three
Standards lin slope. L/D-AAs with short analysis time (< 25 min) and current below 150
µA.
Instrumental precision was tested to check the constancy of The sample volume injected in the capillary influences the
instrumental response to a given analyte in the mid-range of the analytical resolution and sensitivity. To optimize the sample
calibration curve. It was evaluated by multiple injections, n=10 of a volume, three different injection times were assayed for the two
homogeneous standard solution. The intra-day precision of the types of bone cells: 5, 15 and 30 s, at 0.5 psi (33 mbar) while other
method was checked by injecting individual preparations of separation parameters remained constant. At 30 s, even though
standards and samples in the mid-range of the calibration curve. the signal increased, peaks got broader and the selectivity was
Intermediate precision was tested in the same way but on a poorer, so 15 s was selected (data not shown).
different day, with buffer and all the reagents freshly prepared. 3. To enhance the sensitivity, especially for the D-enantiomer,
Results and discussion two different samples volumes were tried in the derivatization
process: 20 and 40 µL while keeping constant the ratio sample and
3.1. Method optimization NBD-F volume to guarantee enough excess of the labeling agent.
The best results were achieved with 40 µL.
The samples in this study are mouse osteocytic MLO-Y4 cell
and osteoblastic MC3T3-E1 cells after addition of lysis RIPA buffer
3
M.P. Lorenzo, et al.
3.2. Method validation
A complete validation was performed for the three D- and L-
amino acids including, Ser, Glu, and Asp. A summary of the
validation parameters is shown in Table 2.
(a) Specificity
As shown in Fig. 1, the method is selective for the analysis and
there are no peaks in the RIPA lysis buffer close to the migration
time of the internal standard or the analytes. The sample ion
strength in the RIPA buffer affects the migration of the analytes as
can be seen in the figure.
(b) Linearity and sensitivity
In the linearity study, the relative response of standards fits the
linear model (r > 0.99) for all AAs and no bias was found for L-Asp
and D-Glu but little bias was found for the rest. Although it has
statistical significance, it has no practical consequences as seen in
the accuracy study. The coefficients of the linear regression along
with their confidence interval are listed in Table 2. The LODs for
the D-amino
Table 2
Validation of the CE-LIF method for the chiral analysis of Asp, Glu and Ser in cellular lysates from osteocytes and osteoblasts. Linearity and estimated limit of detection (LOD) (S/N:3).
Linearity study
Journal of Chromatography B 1152 (2020) 122259
= 3). The inter-assay precision ranged from 2.3% to 14.8% for the
D-AAs and from 2.3% to 11.0% for the L-AAs (n = 6). In the
precision study, all CVs were lower than 10.7% (except for the
LLOQ). Thus, it could be considered adequate.
Regarding the matrix effect, the decay in the signal was lower
than
10.0% for L-Asp and L-Ser and below 20.5% for all analytes.
3.3. Quantitation of D- and L-AAs in osteocytes and osteoblasts
lysates
Finally, the validated method was applied to murine bone cells.
For the analysis of L-Asp and L-Glu, the samples were 10-fold
diluted when their normalized areas were out of the calibration
curve. Once diluted, the derivatization was performed following
the same procedure.
By far, the most abundant amino acid was L-Glu and then L-Asp
(Table 4). This is to be expected, as the L-Glu is the main excitatory
neurotransmitter in the central nervous system [2] and L-Asp
shares with Glu its ability of binding to the NMDA [3] receptor,

tm Range Slope Intercept r LOD

(µM) (µM)

D-Asp 21.8 0.25–2.5 21.6 ± 1.2*10−3 −7.4 ± 2.8*10−3 0.995 0.18

D-Glu D-Ser 17.2 0.25–2.5 12.80 ± 0.84*10−3 0.7 ± 2.0*10−3 0.995 0.22
L-Asp L-Glu 12.4 0.25–2.5 23.5 ± 1.0*10−3 −4.5 ± 2.4*10−3 0.997 0.14
L-Ser 21.6 2.5–50.0 16.80 ± 0.52*10−3 0.5 ± 1.3*10−2 −8.0 ± 0.998
16.4 2.5–50.0 0.998
14.18 ± 0.33*10−3 7.8*10−3
12.1 2.5–50.0 0.996
21.39 ± 0.94*10−3 −2.9 ± 2.2*10−2

acids were estimated from the standard deviation of the y-
intercept in their calibration curve, the LODs were in the range of
0.14–0.22 µmol L−1 for D-Ser and D-Glu respectively. Then, the
lower limit of quantitation, LLOQ, of 0.25 µmol L−1 was tested
experimentally for accuracy and precision with real samples.
(c) Accuracy and precision
In the intra-day accuracy study, values ranged from 81.9% to
111.7%, while in the inter-day study were from 88.2% to 116.2%.
At the LLOQ ranged from 80 to 120% for the three D-AAs (Table 3).
These values could be considered adequate for bioanalysis.
Instrumental precision (n = 10) ranged from 3.7% to 6.4% (CV).
The intra-assay precision ranged from 1.8% to 10.9% for the D-
amino acids, and from 2.5% to 10.3% for the L-amino acids (AAs) (n
though with lower efficiency. Although the osteocytes (MLOY4)
have a diminished metabolism, they have levels of L-Glu almost
doubled than osteoblasts (MC3T3). Osteocytes can initiate bone
remodeling through the activation of bone resorption. Glutamate
signaling performs an essential role in this process, regulating the
expression of different transporters, channels, and proteins for
their release, which controls NMDA, AMPA and mGlu receptor,
expressed by osteoblasts [5,13,16].
Glu is also used as a source of energy in certain cell types, such
as neurons, through its transformation in α-ketoglutarate as a
stage of the citric acid cycle [32]. As osteoblasts have a more active
metabolism and higher energy consumption, it is possible that the
lower levels of glutamate stem from a displacement of the
equilibrium between glutamate

4
M.P. Lorenzo, et al. Journal of Chromatography B 1152 (2020) 122259

Fig. 1. Selectivity assay. A: RIPA Lysis buffer (NBD-F) without IS. B: Blank of sample (NBD-F) with IS. C: Standard mix (50 µmol L −1 L-Glu and L-Ser, 25 µmol L−1 D-
Glu and D-Ser, 12.5 µmol L−1 L- and D-Asp). D: Lysate from osteocyte cell MLOY4. E: Lysate from osteoblast cell MC3T3.
Table 3
Validation of the CE-LIF method for the chosen L/D-amino acids, in cell lysates from osteocytes and osteoblasts. Study of the method́s accuracy, precision and
matrix effect.
Intra-day (n = 3) Inter-day (n = 6)

Concentration (µM) Precision CV Accuracy % Precision CV Accuracy % Matrix effect (%) Instrumental precision (n = 10) CV

D-Asp 0.25 10.9 108.1 9.0 116.2 14.5 4.1

0.50 10.5 81.9 14.8 81.9

1.0 1.8 102.4 2.8 103.2

1.5 6.1 103.7 5.8 106.4

2.5 3.9 96.3 6.5 96.7

D-Glu 0.25 10.6 111.7 8.6 104.6 20.5 6.4

0.50 10.7 100.6 7.2 94.9

1.0 4.9 102.5 4.2 104.4

1.5 1.9 98.6 2.3 100.1

2.5 4.2 100.3 3.2 99.8

D-Ser 0.25 9.2 111.1 12.7 99.3 10.1 5.8

0.50 7.8 97.6 7.0 92.9

1.0 8.0 98.5 6.7 101.8

1.5 5.8 100.4 6.7 100.3

2.5 4.8 98.0 4.8 98.7

L-Asp 2.5 8.3 103.6 7.1 103.3 3.7 3.7

5.0 3.9 95.8 4.9 91.9

10.0 4.8 102.3 4.3 95.8

15.0 3.8 99.2 4.9 95.8

25.0 4.4 100.1 3.5 94.8

L-Glu 2.5 4.3 97.5 5.8 99.8 17.4 4.1

5.0 4.2 97.5 7.2 95.0

5
M.P. Lorenzo, et al. Journal of Chromatography B 1152 (2020) 122259
10.0 5.9 97.5 4.2 103.4

15.0 3.0 97.5 2.3 100.0

25.0 8.7 94.1 3.2 96.4

L-Ser 2.5 10.3 91.3 11.0 88.2 5.3 5.5

5.0 9.1 94.8 9.3 93.0

10.0 6.4 98.3 5.9 102.2

15.0 2.5 102.3 4.9 102.7

25.0 6.7 99.6 5.3 98.3

Table 4 be explained through its biological role, as D-Ser regulates bone

Relative quantitation of D-AAs as D-AA/(D + L)AA and concentrations of L/D- remodeling, inhibiting the osteoclastogenesis and being
Asp, L/D-Glu and L/D-Ser in cellular lysates of osteoblasts (MC3T3) and synthesized and released by near osteoblasts [8].
osteocytes (MLOY4). SEM: Standard error of the mean. Statistical analysis by
Even though these results should be further confirmed with
Mann-Whitney U test.
future experiments with primary cultures of bone cells from mice
Concentration SEM p value
and human, they confirm the presence of D-Ser, D-Asp and D-
D-Asp (µM) MC3T3 1.830 0.025 glutamate in bone cells and their relevant role, especially of Glu in
MLOY4 3.020 0.090 cellular signaling development, therefore constituting a
therapeutic target in the treatment of bone diseases. 4.
D-Asp (%) MC3T3 1.90 0.050 Conclusions

MLOY4 3.65 0.10

A rapid and simple method with sensitivities in the range of
L-Glu (µM) MC3T3 293.2 4.9
nmol L−1 has been optimized and validated for chiral analysis of
MLOY4 585.2 14.5 Asp, Glu, and Ser in two different bone cell lysate samples from
osteocytes and osteoblasts. The application of the method
D-Glu (µM) MC3T3 < 0.25 revealed new results for D- amino acids not published until now.
Significant differences were found (p < 0.05) in the levels of L and
MLOY4 3.23 0.24
D Asp and Glu in both cell lines. Osteocytes have the lowest
D-Glu (%) MC3T3 < 0.085 concentration of L-Asp and the highest of D- Asp, L-Glu, D-Glu, and
L-Ser. By far, the highest difference was found for both L and D-
MLOY4 0.55 0.030 glutamate. Under the current lack of knowledge of chiral amino
acids in bone in physiological conditions of health and disease,
L-Ser (µM) MC3T3 45.4 1.8
MLOY4 56.6 1.6
these findings could be extraordinarily useful in the study of the
role of these neurotransmitters in bone-targeted therapies.
D-Ser (µM) MC3T3 3.22 0.08 Funding
MLOY4 2.73 0.18
This work was supported by the Spanish Ministry of Science,
D-Ser (%) MC3T3 6.65 0.29
Innovation and Universities MICINN, co-funded by European
MLOY4 4.59 0.26 Regional Development Fund (FEDER) RTI2018-095166-B-I00, and
Spanish Ministry of Science, co-funded by European Regional
and α-ketoglutarate to the last one. Development Fund SAF2016-80286-R.
As many authors have reported, D-AAs are usually at very low
concentration in biological samples [4,33,34]. Remarkably, there is CRediT authorship contribution statement
no previous data regarding the levels of D-Glu or D-Asp in
osteocytes or osteoblasts lines or skeletal tissue. In our study, even Maria Paz Lorenzo: Methodology, Investigation, Validation,
though the levels of L-Glu are much higher than those of L-Asp, we Formal analysis, Writing - original draft, Visualization. Luis
found that the concentration of D-Glu in osteoblasts was lower Valiente: Methodology, Investigation, Validation, Writing - original
than the LLOQ. When expressed as relative concentrations over draft, Visualization. Irene Buendia: Resources, Writing - original
the total concentration of amino acid (%D-AA = D-AA/(D + L)AA), draft. Arancha R. Gortázar: Conceptualization, Resources, Writing -
D-Glu was lower than 1.0%, while D-Asp ranged from 1.9% to original draft. Antonia Garcia: Conceptualization, Data curation,
3.6%, being higher in osteocytes with statistically significant Supervision, Writing - review & editing.
difference (Mann-Whitney U test). However, the levels of D-Ser
were close to 3.0%, without any statistically significant difference Declaration of Competing Interest
between the two types of cells.
It is worth mentioning that the lowest concentration of D-Glu The authors declare that they have no competing interests
in osteoblasts matches with the lowest levels of D-Asp and
besides, the L- forms follow the inverse trend. As D-Asp is References
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