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Review

Enantioselective Two-Dimensional High-Performance Liquid
Chromatographic Determination of Amino Acids; Analysis and PhysiologicalSignificance of D-Amino Acids in Mammals
Yurika MIYOSHI, Tsubasa OYAMA, Yusuke ITOH, Kenji HAMASE *Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Abstract
D-Amino acids are now increasingly recognized as physiologically functional molecules and biomarkers in mammals.
However, the amounts of D-amino acids are extremely low in most cases, thus highly sensitive and selective analyticalmethods are practically essential. In the present review article, enantioselective two-dimensional high-performance liquid
chromatographic (2D-HPLC) methods are introduced and their applications to the determination of D-amino acids inmammalian samples are described. For the sensitive determination, amino acid enantiomers are derivatized with
4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and are determined by a fluorescence detector. The 2D-HPLC system
consists of the reversed-phase non-enantioselective, but chemoselective separation of NBD-amino acids in the first dimension,and the sequential chiral separation in the second dimension. By using the system, neuroactive D-amino acids (D-Ser and
D-Ala) were determined in the brain of various mammalian species and the regulation of their intrinsic amounts are also
demonstrated. The physiological and diagnostic meanings of the trace amounts of D-Ser are also discussed. The 2D-HPLC
systems for the acidic D-amino acids (D-Asp and D-Glu), proline analogues (D-Pro, trans- and cis-4-hydroxy-D-Pro) andhydrophilic D-amino acids (D-Arg, D-Asn, D-Asp, D-Gln, D-Glu, D-His, D-Ser, D-allo-Thr and D-Thr) are also shown.
Keywords: D-Amino acids; Enantiomer analysis; 2D-HPLC


1. Introduction
Along with the progress in analytical technologies,
several D-amino acids of the free form and D-amino acidresidues in the proteins were found in higher animals
including humans [1-3]. Free D-amino acids are now
increasingly recognized as novel physiologically active
molecules in the central nervous system and endocrine
tissues, and are also demonstrated to be the biomarkers of
the pathophysiological conditions [4-6]. D-Amino acidresidues in the proteins are shown to have a close relation toaging and also to various diseases [3]. In the present
review article, we focus on the free D-amino acids in thehigher animals, especially in mammals. Concerning theD-amino acid residues in the proteins, please refer to thedetailed articles already published elsewhere [3,7,8].
In mammals, incredibly high amounts of free D-aminoacids were found in specific cases; e.g., D-Ser in the*Corresponding author: Kenji HAMASE
Tel: +81-92-642-6598; Fax: +81-92-642-6598
E-mail: hamase@phar.kyushu-u.ac.jp

cerebrum (30% of the L-Ser [9]) and D-Asp in the pineal
gland (almost the same as or higher than L-Asp [10]).
These D-amino acids are now revealed as a modulator of
the N-methyl-D-aspartic acid (NMDA) subtype ofglutamate receptors (D-Ser [6]), and a regulator of
melatonin secretion (D-Asp [5]). However, the amounts offree D-amino acids in mammalian tissues and physiologicalfluids are extremely low in most cases, and sensitive andselective analytical methods are practically essential.
Enantioselective and two-dimensional high-performanceliquid chromatography (2D-HPLC) is one of the mostpowerful techniques for the determination of trace amountsof D-amino acids in real world samples [2,11]. In thepresent focusing review, 2D-HPLC analysis of D-aminoacids in mammals and the biological significance are
discussed.

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second dimension. As an enantioselective column, aPirkle-type column (Sumichiral OA-2500S, [17,18]) wasused. In the rat plasma, trace amounts of D-Ser and D-Ala
were clearly determined without severe interference byintrinsic compounds in the tissues. The detection limit of
D-Ser using the 2D-HPLC system is 500 amol / injection,and that for D-Ala is also 500 amol / injection.

Fluorescenceintensity

COOH

H

C

NH2

N

+

N

R

C

NH

R

O

N

N

NO2

O

Derivatization of amino acids with NBD-F.

Injector

P
Mobilephase

Micro-ODS
column
1st dimension

Waste

R

D

R

D

2nd dimension
Enantioselective
column

Waste
HPV

P
Loop

20

(b)

Mobilephase
Fig. 2. Flow diagram of the 2D-HPLC system. P: pump, D:
detector, R: integrator, HPV: high pressure valve.

40
L-Ser

60

(c)

(min)
L-Ala

D-Ser
D-Ala

10

20

0

15 (min)Fig. 3. 2D-HPLC chromatograms of D-Ser and D-Ala in rat
plasma.
(a) Reversed-phase separation of NBD-Ser and
NBD-Ala in the first dimension, and the fractions indicated byclosed bars are introduced to the second dimension. (b)Enantiomer separation of NBD-Ser in the second dimension and(c) enantiomer separation of NBD-Ala. Reproduced from ref.
[11] with permission.

NBD-D-Amino acids

For the 2D-HPLC determination of D-Ser and D-Ala inmammals, amino acid enantiomers are derivatized with
4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F [16]) prior
to the HPLC determination (Fig. 1). The fluorescent
NBD-derivatives are first fractionated by a microbore
reversed-phase column as a first dimension, and areautomatically introduced into an enantioselective column
representing the second dimension where the D- andL-enantiomers are separated. The flow diagram of the
2D-HPLC system [13,14] is shown in Fig. 2, and the typicalchromatograms obtained using a biological sample (plasmaof Wistar rat) are described in Fig. 3. In the first
dimension, the fractions of NBD-Ser and NBD-Ala werecollected as their D plus L mixtures by a capillarymonolithic-ODS column, and the enantiomers were further
separated by a narrowbore enantioselective column in the

NBD-AlaNBD-Ser

0

NO2

NBD-F

D-Amino acids

Fig. 1.

H

F

COOH

(a)

0

Fluorescenceintensity

2. Enantioselective 2D-HPLC analysis of Ser and Ala inmammals
D-Ser and D-Ala bind to the glycine site of NMDA
receptors in the central nervous system, and are indicated asco-agonists of the receptor together with L-Glu [6]. These
two D-amino acids modulate the neurotransmission of the
Glu receptors, and novel therapies of neuronal diseases
focusing on these two D-amino acids are expected.
Therefore, the distribution of D-Ser and D-Ala in mammals
and their enzymatic regulation should be clarified in detail.
Although the amount of D-Ser in the frontal brain area isincredibly high, the amounts in the other brain areas andperipheral tissues are low [9,12-14]. The amounts of
D-Ala are also at trace levels in all tissues [13-15].
Therefore, a sensitive and selective determination isnecessary, and for that purpose, 2D-HPLC is a reliable anduseful technique.

2.1. Distribution of D-Ser and D-Ala in mammals
By using the 2D-HPLC system described above, thedistribution of D-Ser in mammals was clarified [13-15].
As experimental animals, rats and mice were used. The
amounts of D-Ser in the central nervous system, peripheral
tissues and physiological fluids are shown in Fig. 4(a). In
the brain, large amounts of D-Ser were found in the cerebral
cortex and hippocampus (the values of %D, (D/(D+L) x100), was around 20-25% for both the rat and mouse),followed by hypothalamus and olfactory lobe. In the
cerebellum and medulla oblongata, the amounts of D-Ser
were trace levels (%D was about 1% or lower). Comparedto the amounts in the frontal brain areas, the amounts of
D-Ser in the peripheral tissues are not significantly high(%D is 0.1-6%). Concerning the physiological fluids, theamount of D-Ser is low in the plasma (%D is 1-2%),
however, the amounts are relatively high in the urine (%Dis 10-50%).
The distributions of D-Ala in the rat and mouse aresummarized to Fig. 4(b). In the brain, the amounts of
D-Ala are low for both rodents (%D is lower than 0.5%),
especially in the hindbrain areas such as cerebellum andmedulla oblongata. On the other hand, the amounts in thepituitary gland and pancreas are relatively high. The

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(a)

400

50

5.0

100

D-Ser
nmol / g or mL

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200

25

2.5

50

0

0
OLF
D-Ala
nmol / g or mL

(b)

CER

HIP

HYP

CEL

MED

0
PIT

PAN

LIV

0
PLA

KID

URI

15

150

15

15

150

10

100

10

10

100

5

50

5

5

50

0

0
OLF

CER

HIP

HYP

CEL MED

0

0
PIT

PAN

LIV

KID

0
PLA

URI

Fig. 4. Distribution of D-Ser (a) and D-Ala (b) in the brain and peripheral tissues. Open bars indicate the values for mice, and closed bars
indicate those for rats. OLF, olfactory bulb; CER, cerebral cortex; HIP, hippocampus; HYP, hypothalamus; CEL, cerebellum; MED, medulla
oblongata; PIT, anterior pituitary; PAN, pancreas; LIV, liver; KID, kidney; PLA, plasma; URI, urine.㻌

highest amount of D-Ala was observed in the pituitary
gland of the rat (%D is 1.3%), and in the pancreas of themouse (%D is 2.7%). The concentration (%D) of D-Ala in
the plasma is about 3% for both animals, however, the %D
is over 15% in the urine.
2.2. Enzymatic regulation of D-Ser and D-Ala inmammalian tissues and physiological fluids
As described above, both D-Ser and D-Ala are noted as
novel candidate biomarkers of neuronal diseases, and thenovel therapies focusing on these two D-amino acids arealso expected. Therefore, it is highly required to clarify
the regulation mechanisms and their contributions on the
intrinsic amounts of D-Ser and D-Ala in mammals. As adegradation enzyme, D-amino acid oxidase (DAO, EC
1.4.3.3) is widely known [1,19,20] and both rat and mousestrains lacking DAO have been established. As a mutant
mouse strain lacking the DAO activity, the ddY/DAOmouse [21] was used, and the LEA/Sen rat was used as amutant rat lacking DAO [22]. As the control mouse, theddY/DAO+ mouse having a normal DAO activity was used,
and the Wistar rat was used as the control rat. By using
the 2D-HPLC system described above, the amounts of
D-Ser and D-Ala in the tissues and physiological fluids ofthese rodents were determined in detail. As shown in Fig.
5(a), the amounts of D-Ser are almost the same in thefrontal brain areas for all the four strains of rodents; i.e.,around 300 nmol of D-Ser / g tissue was observed in the
cerebrum and hippocampus. However, in the hind brain
areas, the amounts of D-Ser in the mutant rats and mice
were drastically higher than those in the control rats andmice. This is because high DAO activity is present in thehind brain areas such as cerebellum and medulla oblongata[23-25] and most of the D-Ser was metabolized by the

enzyme in these tissues. In the peripheral tissues andphysiological fluids, the amounts of D-Ser in the mutant
animals are significantly higher than those in the control
animals in most cases.
The amounts of D-Ala in the ddY/DAO+ mouse,
ddY/DAO- mouse, Wistar rat and LEA/Sen rat are shown in
Fig. 5(b) [13,14,26]. In all the brain areas, peripheral
tissues and physiological fluids, the amounts of D-Ala weredrastically higher in the mutant rodents than those in thecontrol mice and rats. In the control rats, a high DAO
activity is present in the hind brain areas, kidney and liver,and in the control mice, a high DAO activity is present onlyin the hind brain areas and kidney [27]. However, as
shown in Fig. 5(b), the amounts of D-Ala are high in all thetested tissues and physiological fluids regardless of thepresence of DAO. This is mainly due to the difference inthe D-Ala amounts in the serum/plasma. Because theamounts of D-Ala in the blood are low in the control
rodents due to the strong DAO activity in the kidney, theamounts of blood D-Ala are extremely high in the mutant
rodents compared to their control animals. D-Ala present
in the blood immediately distributed to the tissues [28],therefore, the D-Ala amounts in all the tissues could be
upregulated by the decrease in the DAO activity.
As an enzyme synthesizing D-Ser in mammals, Serracemase (SRR, EC 5.1.1.18) has been reported [29,30],
and a knock-out mouse strain lacking SRR (SRR-/- mice)has successfully been established [31-34]. Mice withvarious SRR activities have also been established (SRR-/mice, SRR+/- mice and their control SRR+/+ mice), and theamounts of D-Ser in the entire body of these animals were
determined in detail [34]. The amounts of D-Ser in the
SRR-/- mice were low in all the tested tissues andphysiological fluids, especially, a drastic alteration was

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D-Ser
nmol / g or mL

(a)

450

DAO+ mouse
DAO- mouse

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400

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䠆

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200

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OLF

D-Ala
nmol / g or mL

600

䠆䠆

䠆䠆
䠆䠆

0

(b)

䠆䠆
䠆䠆

100

150

30
䠆䠆

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300

600

150

Wistar rat
LEA/Sen rat

CER

HIP

HYP CEL MED

450

PIT

PAN

䠆䠆

300

KID

PLA

6000

1500
䠆䠆

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300

䠆䠆

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1000

6000

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4000
䠆

䠆䠆
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䠆䠆
䠆䠆

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䠆䠆

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500

2000

䠆䠆

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䠆䠆

HIP

HYP CEL MED

0
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2000
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OLF

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150

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0

LIV

PAN

LIV

0
KID

0
PLA

URI

Fig. 5. Alteration of D-Ser (a) and D-Ala (b) amounts in the brain and peripheral tissues of rodents by the lack of DAO activities. OLF,
olfactory bulb; CER, cerebral cortex; HIP, hippocampus; HYP, hypothalamus; CEL, cerebellum; MED, medulla oblongata; PIT, anterior
pituitary; PAN, pancreas; LIV, liver; KID, kidney; PLA, plasma; URI, urine.

observed in the brain. Fig. 6 shows the results obtained inseveral brain areas, serum and cerebrospinal fluid. By
decreasing the SRR activity, the amounts of D-Ser in mostof the brain areas decreased to around 10% of those in thecontrol mice. These results indicated that the amounts of
D-Ser in the tissues could be regulated by changing the SRR
activity.

D-Ser (nmol / g or mL)

400

**
** **

40
**
** **

300

30

200

20
**
*

100
0

10

CER

HIP

0

CEL

**
* *

SPI

**
*

SER

CSF

Fig. 6. Alteration of D-Ser amounts in the brain and peripheral
tissues of rodents by the lack of SRR activities. Open bars indicate
the values for SRR+/+ mice, gray bars indicate those for SRR+/mice and closed bars indicate those for SRR-/- mice. Reproduced
from ref. [34] with permission. CER, cerebral cortex; HIP,
hippocampus; CEL, cerebellum; SPI, spinal cord; SER, serum;
CSF, cerebrospinal fluid.

2.3. Biological significance of trace amounts of D-Ser inmammalian brain
In the cerebrum and hippocampus of mammals, large
amounts of D-Ser were observed, and the biosynthesis andphysiological function of D-Ser in these tissues have been
investigated in detail [6,35]. On the other hand, theamounts of D-Ser in the hindbrain areas, such as the
cerebellum and medulla oblongata, are extremely low in theadult tissue [13,26,36].
Therefore, the biologicalsignificance of D-Ser in these hindbrain areas has not beenstudied for a long time. Especially, in the cerebellum, asignificant amount of D-Ser is present only during theimmature period [12], and the physiological role of thisD-amino acid in the cerebellum has been a matter of interest.
In 2011, Kakegawa et al. reported that D-Ser plays a crucialrole in regulating the cerebellar long-term depression at thesynapses between parallel fibers and the Purkinje cells [37].
By using the 2D-HPLC system described above, the releaseof D-Ser in the cerebellum of the immature mouse has beenclearly demonstrated.
The amounts of D-Ser are changed in several neuronaldiseases. D-3-Phosphoglycerate dehydrogenase (Phgdh;
EC 1.1.1.95) is an enzyme catalyzing the formation of
3-phosphohydroxypyruvate in the first step of the L-Ser
biosynthesis [38]. The lack of activity of this enzyme
causes severe neurological symptoms as well as a drastic
decrease in the L-Ser amounts. Because D-Ser in the brain
is synthesized from L-Ser by SRR, the amount of D-Ser isalso likely to be decreased. In the brain of the conditional
Phgdh knock-out mouse, the amount of D-Ser has decreased
and only 10% of that in the control mouse was observed
D-Ser is also expected as a biomarker of[39].
amyotrophic lateral sclerosis (ALS).
ALS is aneurodegenerative disorder with a severe loss of
motoneurons in the spinal cord [40,41]. It is a progressive
and fatal disease, and an effective biomarker is highly
needed. Sasabe et al. determined trace amounts of D-Ser
in the spinal cord of the ALS model mouse by the
2D-HPLC system [42]. Compared with the preonset stageof ALS, the amount of D-Ser in the spinal cord has
increased in the onset and terminal stages of the ALS.
These results clearly indicate that trace amounts of D-amino

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acids also have the biological and clinical significance inmammals.
3. Enantioselective 2D-HPLC analysis of Asp and Glu inmammals
D-Asp is widely present in the endocrine tissues ofmammals, and it has been revealed to have physiological
functions in regulating hormonal secretion [5]. D-Glu isalso shown to be present in the urine of various animals[43,44]. In addition to be a regulating molecule ofhormonal secretion, D-Asp and D-Glu have effects onneuronal transmission [4], and therefore, the analysis ofthese two D-amino acids in mammalian tissues andphysiological fluids is highly required. As a sensitive andselective method, a 2D-HPLC system combining a
microbore ODS column and a narrowbore enantioselective
column has been established after derivatization of the Asp
and Glu enantiomers with NBD-F [45].
For thereversed-phase separation of NBD-Asp and NBD-Glu, along microbore monolithic ODS column (0.53 mm i.d. x
750 mm) was used, and a Pirkle-type enantioselective
column (Sumichiral OA-2500S, 1.5 mm i.d. x 250 mm) wasused for their chiral separations. By using the 2D-HPLC
system, trace amounts of D-Asp and D-Glu (500 amol)
could be determined in biological matrices without any
severe interference by intrinsic substances. As the acidic
Table 1. Amounts of D-Asp and D-Glu in the rat tissues andphysiological fluids.
Tissues/fluids

Asp

Glu

Brain Tissues
Cerebral cortex
Hippocampus
Hypothalamus
Cerebellum
Medulla oblongata
Pineal gland
Pituitary gland

12.8㼼1.1 (0.18)
6.0㼼0.6 (0.16)
15.0㼼1.9 (0.32)
28.4㼼3.1 (0.60)
6.5㼼0.8 (0.09)
2025.9㼼147.8 (23.9)
132.5㼼32.3 (3.34)

1.9㼼0.1 (0.04)
3.9㼼0.5 (0.05)
3.2㼼1.0 (0.08)
4.1㼼0.3 (0.15)
2.3㼼0.4 (0.10)
7.2㼼1.1 (0.05)
2.6㼼1.0 (0.05)

Peripheral tissues
Spinal cord
Adrenal gland
TestisThymus
Pancreas
SpleenLung
Heart
KidneyLiver
Muscle6.7㼼0.5 (0.09)
574.3㼼231.2 (70.5)
129.6㼼14.1 (17.9)
88.1㼼0.5 (1.15)
19.3㼼1.6 (0.41)
195.6㼼17.3 (3.61)
141.1㼼13.6 (3.99)
20.2㼼4.3 (1.05)
34.1㼼6.6 (0.43)
14.5㼼3.0 (0.98)
3.5㼼0.5 (1.13)

3.1㼼0.4 (0.08)
0.7㼼0.1 (0.05)
2.0㼼0.4 (0.03)
9.0㼼1.3 (0.09)
1.8㼼0.4 (0.01)
4.4㼼0.6 (0.05)
1.6㼼0.3 (0.02)
0.8㼼0.1 (0.01)
2.8㼼0.5 (0.01)
2.3㼼0.8 (0.03)
1.9㼼0.3 (0.12)

Physiological fluids
PlasmaUrine

0.9㼼0.2 (2.92)
4.1㼼0.9 (10.2)

trace (-)
3.7㼼1.0 (3.33)

Values represent mean ± SE (nmol/g or mL) of 3 rats. Values inparentheses represent %D (D / (D + L) x 100). Rat; Wistar, male, 6
weeks of age. Reproduced from ref. [45] with permission.

amino acid analogs, NMDA, NMLA, N-methyl-D-glutamic
acid (NMDG) and NMLG are also able to be determined by
the 2D-HPLC system with slight modification of theseparation conditions [46].
3.1. Distribution of D-Asp and D-Glu in mammals
Detailed distributions of D-Asp and D-Glu in the brain
and peripheral tissues of the rat have been clarified [45].
The amounts of D-Asp and D-Glu in 7 brain tissues, 11
peripheral tissues, plasma and urine are summarized inTable 1.
Concerning Asp, large amounts of the
D-enantiomer were observed in the endocrine tissues, suchas the pineal gland, pituitary gland, adrenal gland and testis.
However, the amounts of D-Asp in most of the central
nervous system, such as the cerebrum, hippocampus andcerebellum, were trace levels (the values of D-form werelower than 1% of total the Asp in the corresponding tissues).
On the other hand, the amounts of D-Glu are extremely low
in all the tissues tested regardless of the function of thetissue (endocrine tissues or neuronal tissues).
Theamounts of D-Glu are much lower than those of the
L-enantiomer, and the %D values are around 0.1%. Onlyin the urine, a relatively high amount of D-Glu wasobserved, and the %D was 3.3%.
3.2. Relationship between D-Asp amount in the pineal glandand melatonin content
D-Asp was reported to inhibit melatonin secretion in
mammals [47,48].
Melatonin is an indole hormone
synthesized in the pineal gland of mammals [49] mainly byserotonin N-acetyltransferace (SNAT). In rodents, several
strains of mice are known to have a low enzymatic activityof this enzyme, and the amounts of melatonin are extremely
low in the pineal gland of these animals [50,51].
Therefore, it is a matter of interest whether the amounts of
D-Asp in the pineal gland of these mice are related to theamounts of melatonin. In the pineal gland of 3 strains ofrats and 6 strains of mice, both D-Asp and melatonin were
determined [52]. For the determination of D-Asp, a
2D-HPLC system was used, while a pre-column oxidation
column-switching HPLC system was used for thedetermination of melatonin [53].
The results are
summarized in Fig. 7. In all the tested rats, high levels of
D-Asp (almost the same amounts as those of L-Asp) wereobserved, and high levels of melatonin were also present.
On the other hand, in the pineal gland of mice, the amountsof melatonin were totally different between the strains. In
the C3H and CBA strains in which a high SNAT activity
was observed [50], large amounts of melatonin wereobserved in the pineal gland. However, in the pineal
glands of ICR, BALB/c, C57BL and ddY mice, the amountsof melatonin were trace levels. Interestingly, the amountsof pineal D-Asp in the ICR, BALB/c, C57BL and ddY mice

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300

3000

high amounts of this D-amino acid were observed in thepancreas (2.0%) and kidney (8.6%). On the other hand,incredibly high amounts of D-Pro were observed in theurine of both strains of the mouse; the value was 25.9% of
the total Pro in the ddY/DAO+ mouse and 95.1% in the
Especially, in the urine of the
ddY/DAO- mouse.
ddY/DAO- mouse, the amount of D-Pro was almost 20
times higher than that of L-Pro.

0

0

Table 2. Distribution of D-Pro in the tissues and physiologicalfluids of ddY/DAO+ and ddY/DAO- mice.

D-Asp (pmol / gland)
melatonin (fmol / gland)

900

(a)

9000

(b)

60
40

600

20

6000

0

Tissues/fluids

Fig. 7. Amounts of D-Asp and melatonin in the pineal glands ofrats and mice. Closed bars indicate the amounts of D-Asp and open
bars indicate those of melatonin. Reproduced from ref. [52] withpermission.

were also low, while those in the C3H and CBA strains
were high. The amounts of D-Asp in other endocrine
tissues and the plasma of these rats and mice were notdifferent between the strains.
4. Enantioselective 2D-HPLC analysis of Pro analogues
in mammals
D-Pro is one of the D-amino acids widely observed in theplasma and urine in mammalian species [54-56]. The
relationship between plasma D-Pro and a renal disorder was
reported [54,57], and orally or intraventricularly
administered D-Pro affected the hepatic and renal functions[58,59]. Therefore, this D-amino acid is expected as anovel candidate biomarker. A large part of Pro in the
mammalian body is present in the collagen, one of the mostabundant proteins in the mammalian body, and about halfof the Pro in the collagen is converted to hydroxyproline(Hyp).
Therefore, a 2D-HPLC method for theenantioselective determination of Pro and Hyp as theirfluorescent NBD-derivatives has been established [60].
After the reversed-phase separation as the first dimension,enantiomers of the Pro analogues were separated by a
Pirkle-type
enantioselective
column(Sumichiral
OA-2500R) or by a prototype anion-exchange type
enantioselective column (Chiralpak QN-2-AX). By using
these columns, the enantiomers of Pro, trans-Hyp andcis-Hyp were nicely separated in about 10 min.
4.1. Distribution of D-Pro in mammals
By using the 2D-HPLC system, the distributions of D-Pro
both in the ddY/DAO+ mouse and ddY/DAO- mouse havebeen investigated [61,62]. These values are summarizedin Table 2. In the 7 brain areas and 11 peripheral tissues,
trace amounts of D-Pro are found (%D is lower than 1%) in
the ddY/DAO+ mice. In the ddY/DAO- mice, the amountsof D-Pro are generally not very high, however, relatively

ddY/DAO+

ddY/DAO-

Brain Tissues
Cerebral cortex
Cerebellum
Medulla oblongata
Spinal cord
Pineal glanda
Anterior pituitarya
Posterior pituitarya

0.52㼼0.10 (0.25)
0.45㼼0.06 (0.38)
0.30㼼0.01 (0.19)
0.50㼼0.07 (0.36)
3.03㼼0.53 (0.71)
5.13㼼0.83 (0.23)
2.84㼼0.71 (0.43)

0.54㼼0.02 (0.14)
1.26㼼0.06 (1.09)
0.95㼼0.02 (0.82)
0.79㼼0.02 (0.75)
3.86㼼0.79 (1.04)
5.53㼼0.26 (0.30)
1.98㼼0.52 (0.50)

Peripheral tissues
Thyroid gland
Adrenal gland
TestisThymus
Pancreas
SpleenLung
Heart
KidneyLiver
Muscle1.81㼼0.18 (0.47)
1.62㼼0.23 (0.61)
2.87㼼0.18 (1.09)
1.18㼼0.19 (0.21)
2.50㼼0.42 (0.44)
1.39㼼0.14 (0.18)
1.02㼼0.18 (0.51)
0.86㼼0.09 (0.63)
0.45㼼0.06 (0.09)
1.13㼼0.21 (0.19)
1.60㼼0.20 (0.58)

1.97㼼0.38 (0.64)
1.74㼼0.06 (0.83)
3.73㼼0.04 (1.24)
2.21㼼0.43 (0.49)
11.92㼼2.50 (2.03)
3.35㼼0.59 (0.39)
3.15㼼0.42 (1.77)
2.60㼼0.21 (1.67)
19.83㼼4.81 (8.62)
2.39㼼0.29 (0.80)
2.78㼼0.20 (1.71)

Physiological fluids
PlasmaUrine

0.70㼼0.15 (0.68)
23.19㼼5.50 (25.94)

2.69㼼0.47 (3.55)
433.11㼼43.35 (95.09)

Values represent mean ± SE (nmol/g or mL) of 4 mice. aTheamounts of D-Pro are shown as pmol/whole tissue, because thewet weights of these tissues are about 1 mg or lower. Values inparentheses represent %D (D / (D + L) x 100). Reproduced fromref. [61] with permission.

4.2. Origin of D-Pro in mammalian urine
In order to clarify the origin of large amounts of D-Pro
observed in the urine of the ddY/DAO- mouse, the effects
of diet and intestinal bacteria have been investigated [61].
The ddY/DAO- mice were starved for 4 days (only watercontaining 5% sucrose was allowed), and the amounts ofurinary D-Pro were determined. As a result, the amount of
D-Pro gradually decreased, however, a substantial amountof D-Pro was still observed after starvation for 4 days.
The effect of intestinal bacteria was studied using
The
ddY/DAO- mice having a germ-free status.
ddY/DAO mice inoculated with two strains of bacteria(di-associated),andfourstrainsofbacteria(tetra-associated) were also used. In the urine of thegerm-free mice, a large amount of D-Pro was also present,
and the amounts of D-Pro did not change after the

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Fluorescenceintensity(a) 1D: microbore-monolithic ODS column

His

Ser Gln Arg

Asn

Asp Gly

L

His

Ser

Gln

L
D

15

30

Asp

L

0

10

0

10

10 0

Arg
D

10 0
L

Glu

10

0

10

10

L
20

Thr

L

D

D

D
40 0

L
D

0

allo-Thr

Gly

D
20

60 (min)40

Asn D L

D

0

Glu
Thr

0
20
(b) 2D: narrowbore-enantioselective column

0

alloThr20

010Fig. 8. 2D-HPLC chromatograms of hydrophilic D-amino acids in rat urine. (a) Reversed-phase separation of NBD-amino acids in thefirst dimension, and the fractions indicated by gray bars are introduced to the second dimension. (b) Enantiomer separation of NBD-aminoacids in the second dimension. Reproduced from ref. [63] with permission.

inoculation of the bacteria.
These results strongly
indicated that a part of D-Pro was derived from the diet, andalso indicated that the rest of the non-negligible amount
was possibly produced in the mouse.
5. Enantioselective 2D-HPLC analysis of hydrophilicamino acids
As chiral amino acid metabolomics studies gained
attention for the screening of novel physiologically active
substances and biomarkers, several 2D-HPLC procedures
for multiple amino acid enantiomers have been established[63,64]. For the determination of hydrophilic amino acids,
Arg, Asn, Asp, Gln, Glu, His, Ser and Thr were selected as
the targets.
An achiral amino acid, Gly, and astereoisomer of Thr, allo-Thr, were also determined. Afterthe derivatization with NBD-F, these 10 amino acids wereisolated using a long monolithic microbore ODS column,
and the enantiomers were separated by a narrowbore Pirkle
type enantioselective column (Sumichiral OA-2500S)
followed by the determination using a fluorescence detector[63].
Fig. 8 shows the chromatograms of the 10hydrophilic amino acids in the rat urine obtained by theenantioselective 2D-HPLC system. As shown in thechromatograms, all of the D-isomers of the selectedhydrophilic amino acids were present in the rat urine.
Especially, the amounts of D-Asn, D-Ser, D-Arg andD-allo-Thr were relatively high.
6. Enantioselective 2D-HPLC analysis of branchedaliphatic amino acids

Branched aliphatic amino acids (Val, allo-Ile, Ile andLeu) are useful biomarkers of metabolic disorders of aminoacids [65,66]. For the simultaneous determination of thesehydrophobic amino acid enantiomers, a chiral 2D-HPLC
system has been established [64]. A reversed-phase HPLC
was used for the first dimensional separation of the
NBD-derivatives of these amino acids. However, theseparation of allo-Ile and Ile was difficult because they are
structurally similar stereoisomers.
The use of
tetrahydrofuran was essential as an additive to the mobile
phase of the reversed-phase HPLC.
For the chiralseparation in the second dimension, a cinchona
alkaloid-based anion-exchange type enantioselective
column (Chiralpak QN-AX, [67]) was used. By using the
CSP, the enantiomers of all the branched aliphatic aminoacids were separated within 10 min. In the rat urine,
drastically high amounts of D-Val (37% of total Val in theurine), D-allo-Ile (almost 100% of allo-Ile), D-Ile (25% ofIle) and a trace amount of D-Leu (3% of Leu) wereobserved.
7. Conclusion
As described in the present article, various D-amino acids
are indeed present in the higher animals, including humans.
Some of the D-amino acids are trace substances in thetissues and physiological fluids, and the 2D-HPLC
procedures are useful for their determination.
Thephysiological roles, diagnostic meanings, origins andregulation of these D-amino acids in mammals are now
being gradually clarified, and further biochemical and

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clinical investigations together with further progress inanalytical technologies are continuously required.
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