Enantioselective analysis of d - and l -amino acids from mouse macrophages using high performance liquid chromatography

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Enantioselective analysis of d- and l-amino acids from mouse macrophages using high performance liquid chromatography Shiro Kato a , Yuki Masuda b , Morichika Konishi b , Tadao Oikawa a,c,∗ a b c

High Technology Research Core, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan Department of Microbiological Chemistry, Kobe Pharmaceutical University, 4-9-1 Motoyamakita-machi, Higashinada-ku, Kobe, Hyougo 658-8558, Japan Faculty of Chemistry, Materials, and Bioengineering, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan

a r t i c l e

i n f o

Article history: Received 2 December 2014 Received in revised form 5 April 2015 Accepted 20 April 2015 Available online xxx Keywords: d-Amino acids Macrophage Immune cell RAW 264.7 cell Enantioselective analysis

a b s t r a c t The intrinsic d-amino acid profile of mouse macrophages extracted from the peritoneal cavity was analyzed using high performance liquid chromatography. Six d-amino acids (d-Asp, d-Ser, d-Ala, d-Leu, d-Gln and d-Lys) were detected in cell lysates of mouse macrophages. The content and the d/d + l ratio differed depending on the type of d-amino acid and were approximately 3.5–22 nmol/g cells, and approximately 1–20%, respectively. The d-amino acid composition of RAW 264.7 cells, which is a model macrophage cell line, was similar to that of the mouse macrophage. These results suggest that macrophages and RAW 264.7 cells with macrophage-like functions have a similar d-amino acid profile. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Various recent studies on d-amino acids have revealed several important physiological functions of d-amino acids in mammals. d-Ser is present in the mammalian brain, and is a co-agonist for the N-methyl-d-aspartate glutamate receptor [1]. d-Asp is involved in the synthesis, and/or the secretion of hormones such as testosterone [2], and is also involved in neurotransmission [3]. In addition, d-Asp has been suggested to play a role in the mammalian fertilization process, but its mechanism remains unknown [4,5]. The d-Ala concentration in rat tissues and fluids changes and is correlated inversely with the levels of insulin in plasma, and is thought to be related to the circadian rhythm in rats [6]. d-Asp and d-Ser are also thought to be involved in certain diseases. The dSer concentration is increased in the spinal cords of patients with amyotrophic lateral sclerosis (ALS) and in mouse models of ALS [7], however, it is decreased in serum and spinal fluid of patients with schizophrenia [8]. In the case of d-Asp, the concentration and d/d + l ratio in seminal plasma and spermatozoa from patients with teratozoospermia and azoospermia were significantly lower than in those

∗ Corresponding author at: Faculty of Chemistry, Materials, and Bioengineering, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan. Tel.: +81 6 6368 0812. E-mail address: [email protected] (T. Oikawa).

of healthy persons [4]. Accordingly, d-amino acids are thought to be important for human health. As is well-known, the immune system is important for mammalian health. The macrophage is a type of white blood cell and plays a role in immunity. It is a phagocytic cell that engulfs cellular debris and foreign agents such as bacteria. Macrophages are distributed in various tissues [9] and play critical roles in various aspects of innate and adaptive immune responses [10]. Despite the significance of macrophages in human health, little is known about the relationship between macrophages and d-amino acids. In this study, we analyzed the d-amino acid content of macrophages extracted from the mouse peritoneal cavity and of RAW 264.7 cells as a model macrophage cell line using high performance liquid chromatography (HPLC), and described the d-amino acid profiles of these cells to understand the role of d-amino acids in immunity. 2. Materials and methods 2.1. Chemicals and reagents Amino acids were purchased from Wako Pure Chemicals (Osaka, Japan) or Watanabe Kagaku Kogyo (Osaka, Japan). Methanol and acetonitrile were from Kanto Kagaku (Tokyo, Japan). o-Phthalaldehyde (OPA), N-acetyl-l-cysteine (NAC) and 1-aminoadamantane (ADAM) were from Wako Pure Chemicals,

http://dx.doi.org/10.1016/j.jpba.2015.04.028 0731-7085/© 2015 Elsevier B.V. All rights reserved.

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and (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) was from Sigma Aldrich (USA). Other reagents were from Wako Pure Chemicals or Sigma Aldrich. 2.2. Mouse macrophages Peritoneal resident macrophages were extracted from C57BL/6N mice (7–8 weeks old, female) without stimulation with PBS containing 0.02% (w/v) EDTA, and were collected by centrifugation (150 × g, 5 min, 4 ◦ C). The cell pellet was washed once with the same buffer. The extracted cell fraction containing macrophages and other immune cells represented by B and T cells was considered a macrophage fraction because macrophages constituted a major component of this fraction (approximately ∼50%). 2.3. Macrophage-like RAW 264.7 cells RAW 264.7 cells were obtained from the Riken cell bank (Tsukuba, Japan). The cells were grown in RPMI1640 medium containing 10% (v/v) fetal bovine serum (Sigma–Aldrich Japan, Tokyo). After cultivation, the cells were collected by centrifugation and washed twice with PBS containing 0.02% (w/v) EDTA according to the method used for collecting mouse macrophages described in Section 2.1. 2.4. Preparation of cell lysates of macrophages and RAW 264.7 cells Cell lysates of macrophages and RAW 264.7 cells were prepared for amino acid analysis as follows: the cells (1 mg) were resuspended in 50 ␮L of 50 mM potassium phosphate buffer, pH 8.0 (KPB) and then subjected to ultrasonication (output 3, duty cycle 30, 60 sec). The suspension obtained (50 ␮L) was mixed with 20% (w/v) trichloroacetic acid (25 ␮L), incubated for 30 min on ice, and centrifuged (15,000 × g, 30 min, 4 ◦ C) to remove protein. The supernatant obtained (75 ␮L) was mixed with 5 M NaOH (6 ␮L) to adjust pH around 8.0 and was subjected to HPLC analysis. 2.5. Chromatographic analysis The HPLC system was obtained from Shimadzu Co. (Kyoto, Japan) and consisted of a degasser (DGU-20A5 ), a high-pressure gradient solvent delivery unit (LC-20AB and LC-20AD), a communications bus module (CBM-20A), an auto sampler (SIL-20AC), a column oven (CTO-20AC) and a fluorescence detector (RF-10AXL ). d- and l-amino acid concentrations of the cell lysates, mouse feed extract and RPMI1640 medium were determined by HPLC using two pre-column derivatization methods. The derivatization methods with OPA and NAC [11], and with FLEC and ADAM [12], were used for detection of Asp, Glu, Ser, Thr, Ala, Tyr, Val, Met, Trp, Phe, Ile and Leu, and for detection of His, Arg, Asn, Gln and Lys, respectively. The amino acids derivatized with OPA and NAC were separated and quantitated using a Develosil ODS-UG-5 column (250 × 6.0 mm i.d., the particle diameter: 5 ␮m, Nomura Chemical Co., Seto, Japan) equilibrated with 50 mM sodium acetate (mobile phase A). The derivatized amino acids were eluted with a gradient of methanol (mobile phase B; linear gradient as follows; 0%, 0 min; 24%, 16–24 min; 40%, 29–50 min; 67%, 69 min; 80%, 69.01–74 min). The flow rate was 1.2 mL/min, and the elution was monitored by fluorescence (excitation at 340 nm, emission at 450 nm). The amino acids derivatized with FLEC and ADAM were analyzed using a GROM-SIL FLEC-1 column (250 × 4.0 mm i.d., the particle diameter: 3 ␮m, Grom Analytik + HPLC, Rottenburg-Hailfingen, Germany). The column was equilibrated with 50 mM sodium

acetate buffer, pH 4.0 (mobile phase A), and acetonitrile (mobile phase B, linear gradient as follows; 22%, 0 min; 30%, 14 min; 36.2%, 26 min; 62%, 26.01 min; 72%, 56 min; 20%, 76 min) and tetrahydrofuran (mobile phase C, linear gradient as follows; 8%, 0 min; 0%, 14–56 min; 8%, 76 min) were used for elution of the derivatized amino acids. The flow rate was 0.75 mL/min, and the elution was monitored by fluorescence (excitation at 263 nm, emission at 313 nm).

3. Results and discussion The amino acid composition of peritoneal macrophages extracted from C57BL/6N mice was analyzed using HPLC. Six damino acids (d-Asp, d-Ser, d-Ala, d-Leu, d-Gln and d-Lys) were detected in the cell lysates of mouse macrophages (Fig. 1A and B). The peaks of d-Asp, d-Ser, d-Ala, d-Leu, d-Gln and d-Lys were overlapped well with those of authentic d-amino acids. The concentrations of d-Asp, d-Ser, d-Ala, d-Leu and d-Lys were decreased when the sample was treated with d-amino acid oxidase (10 ␮L) (DAO (0.1 U/mL), from porcine kidney, Sigma–Aldrich, USA). The peak of d-Gln was detected if the gradient conditions changed. The d- and l-amino acid contents are summarized in Table 1. The d-amino acid content and their d/d + l ratios varied from approximately 3.5–22 nmol/g cells, and approximately 1–20%, respectively. RAW 264.7 is a mouse leukemic monocyte macrophage cell line that exhibits a macrophage-like ability to respond to foreign stimuli. It is widely used as a model cell line to study macrophage function. The d-amino acid composition of RAW 264.7 cells was also analyzed (Fig. 1C and D), and the results are summarized in Table 1. The detected d-amino acids were also analyzed after DAO treatment and were confirmed by co-chromatography according to the same method described above for the macrophage sample. The d-amino acid composition of RAW 264.7 cells was similar to that of the macrophage, suggesting that macrophages and RAW 264.7 cells tend to maintain a similar d-amino acid profile. Based on these results, the RAW264.7 cell line is thought to be a promising model for understanding the function(s) of d-amino acids in macrophages, and the origin of the detected d-amino acids is therefore of great interest. In order to determine the origin of the d-amino acids detected in macrophages and RAW 264.7 cells, we analyzed the d-amino acid contents of mouse feed and RPMI1640 medium used in this study. In mouse feed, d-Asp, d-Ser, d-Ala, d-Leu and d-Lys were detected, and RPMI1640 medium contained d-Asp, d-Ser, d-Ala and d-Lys (Supplementary Table 1). On the contrary, d-Gln was not detected in mouse feed and RPMI1640 medium, and d-Leu was not found in RPMI1640 medium, although these two amino acids were detected in cell lysates of mouse macrophages and RAW 264.7 cells. These results suggest that at least a part of the d-amino acids detected in macrophages and RAW 264.7 cells were endogenously synthesized, and were not derived from an exogenous source. The enzymes serine racemase [13] and aspartate racemase [14], which endogenously synthesize d-Ser and d-Asp, respectively, have been identified in mammals. It has been suggested that the origin of d-Ala in mice is intestinal bacteria [15]. However, the origin of other d-amino acids in mammals remains unclear. Our results raise the possibility that novel amino acid racemase(s) acting on d-Gln and d-Leu exist in mammalian cells. Although the physiological function(s) of d-amino acids in macrophages remains unclear, the present study demonstrated that macrophages contain significant amounts of d-amino acids. To our knowledge, this is the first report describing the analysis of the d-amino acid content of macrophage. In human brain,

Please cite this article in press as: S. Kato, et al., Enantioselective analysis of d- and l-amino acids from mouse macrophages using high performance liquid chromatography, J. Pharm. Biomed. Anal. (2015), http://dx.doi.org/10.1016/j.jpba.2015.04.028

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B

A

0

3

10

20

30 40 50 Retention time (min)

L-Asp

L-Ser

10 11 12 13 14

24

70

26

38

0

10

39

20

40

41

68 69 70 71 72

D-Gln

19

21

30 40 50 Retention time (min)

60

70

80

60

70

80

L-Lys

L-Gln

D-Leu

D-Ala

25

80

L-Leu

L-Ala

D-Ser

D-Asp

60

D-Lys

23

25

52

53

54

D C

0 0

D-Asp (shoulder)

10

20

30 40 50 Retention time (min) L-Ser

L-Asp

60

70

L-Ala

10

20

80

30 40 50 Retention time (min)

L-Leu

L-Lys

L-Gln D-Gln D-Ser

10 11 12 13 14

24

25

D-Leu

D-Ala

26

37 38 39 40

68 69 70 71 72

21

23

25

52 53 54 55

Fig. 1. Chromatograms of mouse macrophages (A and B) and RAW 264.7 cells (C and D) derivatized with OPA/NAC (A and C) or FLEC/ADAM (B and D). Under each chromatogram, enlarged views of the d-amino acid peak regions are shown. Table 1 d- and l-amino acid compositions of mouse macrophages and RAW 264.7 cells. Content (nmol/g cells) Macrophage (n = 3) l-Asp d-Asp l-Ser d-Ser l-Ala d-Ala l-Leu d-Leu l-Gln d-Gln l-Lys d-Lys

719 21.9 227 4.86 328 3.54 121 9.11 66.7 14.5 91.4 b

± ± ± ± ± ± ± ± ± ± ±

164 1.8 75 0.24 18 1.03 28 1.34 37.1 0.9 47.8

d/d + l ratio (%) RAW 264.7 cell (n = 2)

Macrophage (n = 3)

RAW 264.7 cell (n = 2)

375 ± 95

3.1 ± 0.8

Not calculated

2.3 ± 1.0

1.8 ± 0.3

1.1 ± 0.3

5.4 ± 2.5

7.2 ± 0.9

14 ± 1

20 ± 9

53 ± 1

Not calculated

0

a

127 ± 31 2.43 ± 1.00 51.6 ± 2.5 2.92 ± 1.28 61.9 ± 15.0 9.85 ± 1.75 35.1 ± 2.2 40.2 ± 3.4 21.2 ± 3.0 Not detected

The values were shown as mean ± standard deviation. a Could not be separated from the interfering substance. b Below the limit of quantification (LOQ). The LOQ values determined for both enantiomers of Asp, Ser, Ala, Leu, Gln and Lys were 2, 2, 2, 2, 8 and 10 nmol/g cells, respectively.

d-Asp occurs transiently at very high levels during the last stage of embryonic life or in early postnatal life. In the human fetal cortex, the concentration of d-Asp transiently exceeded that of l-Asp [16]. Macrophages normally exist in a resting state, and various stimuli can activate them during an immune response [17,18]. In this study, we analyzed the d-amino acid composition of macrophages at rest, and RAW 264.7 cells without stimulation. Accordingly, further investigations concerning alterations of d-amino acids during an immune response will be required to clarify the physiological functions of d-amino acids in macrophages. DAO oxidatively degrades d-amino acids into corresponding ␣-keto acids and ammonia, and plays an important role in damino acid metabolism in mammals. The presence of the enzyme in macrophages and other monocytes has been reported [19–22], and these investigations have focused on bacterial killing that is

mediated by hydrogen peroxide that is produced by the action of amino acid oxidases. DAO activity has been considered to contribute in part to protection of host cells against bacterial infection [22]. However, the DAO activities reported for mammalian cells were too low to accomplish this function [19–22]. In addition, our preliminary analysis demonstrated that significant DAO activity was not detected in cell lysates of RAW 264.7 cells (data not shown). The variety of d-amino acids detected in macrophages may reflect the low DAO activity present in these cells. Recently, d-Ser was reported to negatively regulate osteoclastogenesis [23]. Osteoclasts are derived from macrophages, and our research, which is focused on d-amino acids in macrophages, is novel and important research. We are currently studying the physiological function(s) of d-amino acids in macrophages in various biological systems.

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4. Conclusion In the present study, the d-amino acid compositions of mouse macrophages and macrophage-like RAW 264.7 cells have been successfully analyzed using HPLC. Our results suggest that conventional HPLC methods are capable of quantifying levels of intrinsic d-amino acids in mouse macrophages, and we expect that the d-amino acid profile of macrophages shown here will provide a basis for future research aimed at understanding the physiological function of d-amino acids in immune cells. Acknowledgment This study was supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) (No. S1311044). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jpba.2015.04.028 References [1] P.D. Leeson, L.L. Iversen, The glycine site on the NMDA receptor: structure–activity relationships and therapeutic potential, J. Med. Chem. 37 (1994) 4053–4067. [2] Y. Nagata, H. Homma, M. Matsumoto, K. Imai, Stimulation of steroidogenic acute regulatory protein (StAR) gene expression by d-aspartate in rat Leydig cells, FEBS Lett. 454 (1999) 317–320. [3] S. D’Aniello, I. Somorjai, J. Garcia-Fernàndez, E. Topo, A. D’Aniello, d-Aspartic acid is a novel endogenous neurotransmitter, FASEB J. 25 (2011) 1014–1027. [4] G. D’Aniello, S. Ronsini, F. Guida, P. Spinelli, A. D’Aniello, Occurrence of daspartic acid in human seminal plasma and spermatozoa: possible role in reproduction, Fertil. Steril. 84 (2005) 1444–1449. [5] G. D’Aniello, N. Grieco, M.A. Di Filippo, F. Cappiello, E. Topo, E. D’Aniello, S. Ronsini, Reproductive implication of d-aspartic acid in human pre-ovulatory follicularfluid, Human Reprod. 22 (2005) 3178–3183. [6] K. Hamase, A. Morikawa, S. Etoh, Y. Tojo, Y. Miyoshi, K. Zaitsu, Analysis of small amounts of d-amino acids and the study of their physiological functions in mammals, Anal. Sci. 25 (2009) 961–968. [7] J. Sasabe, T. Chiba, M. Yamada, K. Okamoto, I. Nishimoto, M. Matsuoka, S. Aiso, d-Serine is a key determinant of glutamate toxicity in amyotrophic lateral sclerosis, EMBO J. 26 (2007) 4149–4159.

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