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Developmental changes in free D-aspartic acid in the chicken embryo and in the neonatal rat
Life Sciences, Printed in the
Vol.
46,
Pergamon
1517-1522
pp.
Press
U.S.A.
DEVELOPMENTAL CHANGESIN FREE D-ASPARTIC ACID IN THE CHICKEN EMBRYOAND IN THE NEONATALRAT Amos Neidle Nathan
S.
Kline
and David
Institute
Orangeburg, (Received
in final
for
S. Dunlop
Research
Psychiatric
New York,
10962
form March 13,
1990)
Summary acid was measured in fertilized chicken Free D-aspartic In each tissue and neonatal rats. eggs, chicken embryos, examined a maximum value was found at a characteristic time of development. For the chicken embryo brain, the maximum was 9% D incubation: for the retina, 20% D at 13 days of at 11 days of
In the neonatal rat, as in the chicken embryo, Dincubation. aspartic acid continued to increase in the retina after ‘that in The maximum, the brain and other tissues had begun to decline. Thus in two I days 29% D, was found after birth. phylogenetically distant species, similar developmental patterns Some data on of D-aspartic acid change were observed. similarities between the D/L aspartic acid ratios of adult In addition, the chicken and rat tissues are also reported. total D-aspartic acid content of the egg, including the embryo, increased from 44 nmol at day 1 to 159 nmol at day 12, showing
that release from continuing process
a bound form or during development.
de
novo
synthesis
is a
Free D-aspartic acid is present in various tissues of rats and mice with particularly high concentrations in the cerebral hemispheres of neonatal the concentration of D-aspartic acid falls rapidly brain. In this region, after birth, from 160 nmol/g (8.4% D) to 13 nmol/g (0.4% D) within 10 days These results suggested that characteristic patterns of change in D(1). aspartic acid levels might take place within the embryo, in regard to both tissue distribution and time of development. Since the chicken embryo provides a convenient, well characterized, system for developmental studies, we have been investigating changes in free D-aspartic acid levels in this organism. One of the observations to be reported is that D-aspartic acid concentration reaches a peak in the retina at day 13 of incubation. Since, in the rat, the corresponding stage of development of the eye occurs postnatally, retina and other tissues from this species were examined, from birth to 17 days of age. In addition, we wish to report similarities in the tissue distribution free D-aspartic acid in the adult rat and chicken, again pointing analogous functional or developmental roles for this amino acid.
0024-3205190 Copyright
(c)
$3.00
+.oo
1990 Pergamon
Press
plc
of to
1518
Developmental Changes in D-Aspartate
Hethods
Vol. 46, No. 21,199O
and Haterials
Biological materials. Fertile white Leghorn chicken eggs were obtained from Avian Services, Frenchtown, PA. The rats studied were from the Sprague Dawley strain. Determination of D-aspartic acid. D-aspartic acid was measured as described previously (1). Chick embryo tissues were homogenized in 9 volumes of water containing l/20 volume of 60% perchloric acid. After centrifugation, the supernatant solution with sodium was neutralized hydroxide and derivatized with naphthylisocyanate. Naphthylcarbamoyl aspartic acid was then isolated using liquid chromatography on Cl0 reversed phase columns and resolved on columns saturated with L-aspartyl Lphenylalanyl methyl ester. For some determinations a Vydak TP CIE column was eluted with a pH 5.7 buffer 5mM in sodium phosphate and 5mM in sodium acetate. The validity of the method was established (1) by comparing its results with those obtained by resolving isolated aspartic acid using naphthylethyl isocyanate (2) or o-phthalaldehyde and N-acetyl L-cysteine (3). Results The method used for the determination of DD-aspartic acid measurement. aspartic acid is based on the prior formation and isolation of the naphthylcarbamoyl aspartic acid derivatives. This preliminary step separates the derivatives from small quantities of interfering substances. Separation of the enantiomers is then accomplished using a Cl@ reversed phase column made asymmetric by equilibration with L-aspartyl L-phenylalanine methyl ester The retentivity and consequently the resolving ability of CIS (FIG. 1).
6.
A.
ELUTION
FIG. Resolution of previously Chicken consecutively.
MINUTES
TIME
1
isolated naphthylcarbamoyl embryo, day 16. A. Retina.
aspartic acid, B. Brain.
run
Vol.
46, No. 21,
columns retention lowering
Developmental
1990
Changes
in D-Aspartate
differs among columns of different manufacture. However, of D- and L-naphthylcarbamoyl aspartic acid can be increased the pH of the phosphate buffer or by increasing its concentration.
1519
the by
Embryological deVelOpBent in the chick. As had been indicated by the rapid postnatal decline in free aspartic acid D/D+L ratios in the rat several tissues in the chicken reached peak ratios cerebral hemisphere, at which these peaks during embryological development (FIG. 2). The times and 13 days of incubation for heart, brain, were at 8, 10, 11, occurred respectively. The extent of maximal D-aspartic acid muscle, and retina, accumulation also differed among different tissues. The highest level was found in retina (20% D) followed by brain (9%), heart (3%), and muscle (2%). of D-aspartic acid were found in liver regardless of incubation Only traces Although we could not obtain accurate fresh weights for the embryo time. the aspartic acid concentration, at least of brain, does not change tissues, much during 8-19 days of incubation (41, suggesting that similar maxima would be seen if D-aspartic acid were expressed as concentration rather than as %D.
CHICK
EMBRYO
0
5
10
15
INCUBATION
FIG.
20
(DAYS)
2
The % of D-aspartic acid (D/D+L x 1001 in various chick embryo tissues as a function of incubation time. Each point represents the average of duplicate determinations of D-aspartic acid in pooled samples of tissue from two or more embryos.
1520
Developmental Changes in D-Aspartate
Vol. 46, No. 21, 1990
Since in the chick embryo, the Some riostnatal chanues in the rat. highest level of free D-aspartic acid in the retina occurs several days later than that in the brain, it seemed possible that, if a similar in the rat, the retina of this species developmental pattern were present We therefore might reach its maximumD/D+L aspartic acid ratio postnatally. measured D-aspartic acid in retina, cerebral hemispheres, and blood in the The D/D+L ratio in the retina three week period after birth (PIG. 3). reached a peak of 29% at seven days. Since the aspartic acid content of the retina is about 1.2 umol/g, 0.35 urn01 of D-aspartic acid/g is present, a For neonatal rat value higher than those of most essential amino acids. tissues, the data were also plotted as Nmoles/g wet weight against age. Curves similar to those for the D/D+L ratios were obtained, indicating that increases in D-aspartic acid concentrations rather than decreases in those of the L-isomer had taken place.
RAT
0
10
5
DAYS
AFTER
15
BlRTH
FIG. 3 Free D-aspartic acid in retina, cerebral hemispheres and blood of rats from birth to 17 days of age. Each point represents the D-aspartic content in tissues from 3 or more embryos. Changes in free D-aSDarth acid in the chicken ega during incubation. In the rat, D-aspartic acid is present in the maternal blood supply (l), making it difficult to determine whether it originates within the embryo during development. Since the chicken egg comprises a closed system, an increase in
Vol.
46, No. 21,
1990
Developmental ChangesinD-Aspartate
1521
the total free D-aspartic acid of the egg during incubation would indicate that either release of D-aspartic acid from precursor molecules or that the synthesis de novo of D-aspartic acid had taken place. At day 1, the Daspartic acid content was 44.5 + 9.2 nmol/egg (0.16 f 0.03% D, n=4). At day 12 the figures were 159 f 30, and 0.6% f O.l%, respectively. The increase has a p value of
D-Aspartic
acid
in adult
Rat* Tissue Blood Plasma Red cells Brain Hemisphere Cerebellum Spinal cord Retina Pituitary Liver Kidney Heart Muscle * **
rat
and chicken
tissues.
Chicken** DJ D+L
(%I
6.2 2.6 1.2
14.2 4.7 18.0
0.43 0.79 0.13 1.5 3.8 1.0 0.5
Except for the retina value, Average of two determinations.
0.64 0.66 1.6 0.5 0.2 0.3 the data
is
from (1).
Discussion Data have been presented indicating that free D-aspartic acid has a similar tissue distribution and developmental profile in both the rat and chicken. These parallel distributions in phylogenetically distant species suggest that such patterns may be widespread among vertebrates. It seems possible that D-aspartic acid may play a functional role in early development, perhaps acting as a regulatory substance, initiating or inhibiting a sequence of developmental events. Such mechanisms could be reflected in the observation that peaks of D-aspartic acid concentration occur in the same order as the development of functional activity (heart, then brain, then retina). Alternatively, the parallel changes that we observe could indicate that there is an obligatory sequence in the development of biosynthetic, degradative, or transport processes.
1522
Developmental Changes in D-Aspartate
Vol. 46, No. 21,lVVO
The source of free D-aspartic acid is not known in either the chicken or however, the total content of D-aspartic acid the rat. In the chicken egg, increased from day 1 to day 12, indicating that D-aspartic acid had either been released from proteins or other precursors initially present within the egg or was synthesized de novo by the embryo. The obvious possibility that D-aspartic acid arises directly from the racemization of free L-aspartic acid appears unlikely since attempts to introduce 14C from glucose into D-aspartic acid have thus far been unsuccessful. In these experiments, with newborn rat in vivo and chick embryo retina substantial quantities of label in vitro, were incorporated into the L-isomer.
acid, which presumably arises from racemization Protein-bound D-aspartic is known to occur in brain white matter (5,6), myelin basic protein in situ, (71, and erythrocyte membranes (8). It seems possible, therefore, that small amounts of D-aspartic acid are present in other proteins as well, and that might contribute to free D-aspartic acid pools. protein turnover in general Other possible mechanisms for the production of free D-aspartic acid such as racemization during protein degradation or racemization as a byproduct of transamination reactions (9) are also being investigated. References 1. 2. 3. 4. 5. 6. 7. 8. 9.
D.S. DUNLOP, A. NEIDLE, D. McHALE, D.M. DUNLOPand A. LAJTHA, Biochem. Biophys. Res. Comm. 141, 27-32 (1986). D.S. DUNLOPand A. NEIDLE, Anal. Biochem. 165, 38-44 (1987). D.W. ASWAD, Anal. Biochem. 138, 405-409 (1984). G. LEVI and G. MORISI, Brain Res. 26, 131-140 (1971). G.H. FISHER, N.M. GARCIA, I.L. PAYAN, R. CADILLA-PEREZRIOS, W.A. SHEREMATAand E.H. MAN, Biochem. Biophys. Res. Comm. 135, 683-687 (19861. E.H. MAN, G.H. FISHER, 1-L. PAYAN, R. CADILLA-PEREZRIOS, N.M. GARCIA, R. CHEMBURKAR, G. ARENDSand W.H. FREY II, J.Neurochem. 48, 510-515 (1987). R. SHAPIRA, K.D. WILKINSON and G. SHAPIRA, J.Neurochem. 49, 649-654 (1988). L.S. BRUNAUERand S. CLARKE, J. Biol. Chem. 261, 12538-12543 (1986). K. SUNIL and P. CHRISTEN, Eur. J. Biochem. 175, 433-438 (1988).
Vol.
46,
Pergamon
1517-1522
pp.
Press
U.S.A.
DEVELOPMENTAL CHANGESIN FREE D-ASPARTIC ACID IN THE CHICKEN EMBRYOAND IN THE NEONATALRAT Amos Neidle Nathan
S.
Kline
and David
Institute
Orangeburg, (Received
in final
for
S. Dunlop
Research
Psychiatric
New York,
10962
form March 13,
1990)
Summary acid was measured in fertilized chicken Free D-aspartic In each tissue and neonatal rats. eggs, chicken embryos, examined a maximum value was found at a characteristic time of development. For the chicken embryo brain, the maximum was 9% D incubation: for the retina, 20% D at 13 days of at 11 days of
In the neonatal rat, as in the chicken embryo, Dincubation. aspartic acid continued to increase in the retina after ‘that in The maximum, the brain and other tissues had begun to decline. Thus in two I days 29% D, was found after birth. phylogenetically distant species, similar developmental patterns Some data on of D-aspartic acid change were observed. similarities between the D/L aspartic acid ratios of adult In addition, the chicken and rat tissues are also reported. total D-aspartic acid content of the egg, including the embryo, increased from 44 nmol at day 1 to 159 nmol at day 12, showing
that release from continuing process
a bound form or during development.
de
novo
synthesis
is a
Free D-aspartic acid is present in various tissues of rats and mice with particularly high concentrations in the cerebral hemispheres of neonatal the concentration of D-aspartic acid falls rapidly brain. In this region, after birth, from 160 nmol/g (8.4% D) to 13 nmol/g (0.4% D) within 10 days These results suggested that characteristic patterns of change in D(1). aspartic acid levels might take place within the embryo, in regard to both tissue distribution and time of development. Since the chicken embryo provides a convenient, well characterized, system for developmental studies, we have been investigating changes in free D-aspartic acid levels in this organism. One of the observations to be reported is that D-aspartic acid concentration reaches a peak in the retina at day 13 of incubation. Since, in the rat, the corresponding stage of development of the eye occurs postnatally, retina and other tissues from this species were examined, from birth to 17 days of age. In addition, we wish to report similarities in the tissue distribution free D-aspartic acid in the adult rat and chicken, again pointing analogous functional or developmental roles for this amino acid.
0024-3205190 Copyright
(c)
$3.00
+.oo
1990 Pergamon
Press
plc
of to
1518
Developmental Changes in D-Aspartate
Hethods
Vol. 46, No. 21,199O
and Haterials
Biological materials. Fertile white Leghorn chicken eggs were obtained from Avian Services, Frenchtown, PA. The rats studied were from the Sprague Dawley strain. Determination of D-aspartic acid. D-aspartic acid was measured as described previously (1). Chick embryo tissues were homogenized in 9 volumes of water containing l/20 volume of 60% perchloric acid. After centrifugation, the supernatant solution with sodium was neutralized hydroxide and derivatized with naphthylisocyanate. Naphthylcarbamoyl aspartic acid was then isolated using liquid chromatography on Cl0 reversed phase columns and resolved on columns saturated with L-aspartyl Lphenylalanyl methyl ester. For some determinations a Vydak TP CIE column was eluted with a pH 5.7 buffer 5mM in sodium phosphate and 5mM in sodium acetate. The validity of the method was established (1) by comparing its results with those obtained by resolving isolated aspartic acid using naphthylethyl isocyanate (2) or o-phthalaldehyde and N-acetyl L-cysteine (3). Results The method used for the determination of DD-aspartic acid measurement. aspartic acid is based on the prior formation and isolation of the naphthylcarbamoyl aspartic acid derivatives. This preliminary step separates the derivatives from small quantities of interfering substances. Separation of the enantiomers is then accomplished using a Cl@ reversed phase column made asymmetric by equilibration with L-aspartyl L-phenylalanine methyl ester The retentivity and consequently the resolving ability of CIS (FIG. 1).
6.
A.
ELUTION
FIG. Resolution of previously Chicken consecutively.
MINUTES
TIME
1
isolated naphthylcarbamoyl embryo, day 16. A. Retina.
aspartic acid, B. Brain.
run
Vol.
46, No. 21,
columns retention lowering
Developmental
1990
Changes
in D-Aspartate
differs among columns of different manufacture. However, of D- and L-naphthylcarbamoyl aspartic acid can be increased the pH of the phosphate buffer or by increasing its concentration.
1519
the by
Embryological deVelOpBent in the chick. As had been indicated by the rapid postnatal decline in free aspartic acid D/D+L ratios in the rat several tissues in the chicken reached peak ratios cerebral hemisphere, at which these peaks during embryological development (FIG. 2). The times and 13 days of incubation for heart, brain, were at 8, 10, 11, occurred respectively. The extent of maximal D-aspartic acid muscle, and retina, accumulation also differed among different tissues. The highest level was found in retina (20% D) followed by brain (9%), heart (3%), and muscle (2%). of D-aspartic acid were found in liver regardless of incubation Only traces Although we could not obtain accurate fresh weights for the embryo time. the aspartic acid concentration, at least of brain, does not change tissues, much during 8-19 days of incubation (41, suggesting that similar maxima would be seen if D-aspartic acid were expressed as concentration rather than as %D.
CHICK
EMBRYO
0
5
10
15
INCUBATION
FIG.
20
(DAYS)
2
The % of D-aspartic acid (D/D+L x 1001 in various chick embryo tissues as a function of incubation time. Each point represents the average of duplicate determinations of D-aspartic acid in pooled samples of tissue from two or more embryos.
1520
Developmental Changes in D-Aspartate
Vol. 46, No. 21, 1990
Since in the chick embryo, the Some riostnatal chanues in the rat. highest level of free D-aspartic acid in the retina occurs several days later than that in the brain, it seemed possible that, if a similar in the rat, the retina of this species developmental pattern were present We therefore might reach its maximumD/D+L aspartic acid ratio postnatally. measured D-aspartic acid in retina, cerebral hemispheres, and blood in the The D/D+L ratio in the retina three week period after birth (PIG. 3). reached a peak of 29% at seven days. Since the aspartic acid content of the retina is about 1.2 umol/g, 0.35 urn01 of D-aspartic acid/g is present, a For neonatal rat value higher than those of most essential amino acids. tissues, the data were also plotted as Nmoles/g wet weight against age. Curves similar to those for the D/D+L ratios were obtained, indicating that increases in D-aspartic acid concentrations rather than decreases in those of the L-isomer had taken place.
RAT
0
10
5
DAYS
AFTER
15
BlRTH
FIG. 3 Free D-aspartic acid in retina, cerebral hemispheres and blood of rats from birth to 17 days of age. Each point represents the D-aspartic content in tissues from 3 or more embryos. Changes in free D-aSDarth acid in the chicken ega during incubation. In the rat, D-aspartic acid is present in the maternal blood supply (l), making it difficult to determine whether it originates within the embryo during development. Since the chicken egg comprises a closed system, an increase in
Vol.
46, No. 21,
1990
Developmental ChangesinD-Aspartate
1521
the total free D-aspartic acid of the egg during incubation would indicate that either release of D-aspartic acid from precursor molecules or that the synthesis de novo of D-aspartic acid had taken place. At day 1, the Daspartic acid content was 44.5 + 9.2 nmol/egg (0.16 f 0.03% D, n=4). At day 12 the figures were 159 f 30, and 0.6% f O.l%, respectively. The increase has a p value of
D-Aspartic
acid
in adult
Rat* Tissue Blood Plasma Red cells Brain Hemisphere Cerebellum Spinal cord Retina Pituitary Liver Kidney Heart Muscle * **
rat
and chicken
tissues.
Chicken** DJ D+L
(%I
6.2 2.6 1.2
14.2 4.7 18.0
0.43 0.79 0.13 1.5 3.8 1.0 0.5
Except for the retina value, Average of two determinations.
0.64 0.66 1.6 0.5 0.2 0.3 the data
is
from (1).
Discussion Data have been presented indicating that free D-aspartic acid has a similar tissue distribution and developmental profile in both the rat and chicken. These parallel distributions in phylogenetically distant species suggest that such patterns may be widespread among vertebrates. It seems possible that D-aspartic acid may play a functional role in early development, perhaps acting as a regulatory substance, initiating or inhibiting a sequence of developmental events. Such mechanisms could be reflected in the observation that peaks of D-aspartic acid concentration occur in the same order as the development of functional activity (heart, then brain, then retina). Alternatively, the parallel changes that we observe could indicate that there is an obligatory sequence in the development of biosynthetic, degradative, or transport processes.
1522
Developmental Changes in D-Aspartate
Vol. 46, No. 21,lVVO
The source of free D-aspartic acid is not known in either the chicken or however, the total content of D-aspartic acid the rat. In the chicken egg, increased from day 1 to day 12, indicating that D-aspartic acid had either been released from proteins or other precursors initially present within the egg or was synthesized de novo by the embryo. The obvious possibility that D-aspartic acid arises directly from the racemization of free L-aspartic acid appears unlikely since attempts to introduce 14C from glucose into D-aspartic acid have thus far been unsuccessful. In these experiments, with newborn rat in vivo and chick embryo retina substantial quantities of label in vitro, were incorporated into the L-isomer.
acid, which presumably arises from racemization Protein-bound D-aspartic is known to occur in brain white matter (5,6), myelin basic protein in situ, (71, and erythrocyte membranes (8). It seems possible, therefore, that small amounts of D-aspartic acid are present in other proteins as well, and that might contribute to free D-aspartic acid pools. protein turnover in general Other possible mechanisms for the production of free D-aspartic acid such as racemization during protein degradation or racemization as a byproduct of transamination reactions (9) are also being investigated. References 1. 2. 3. 4. 5. 6. 7. 8. 9.
D.S. DUNLOP, A. NEIDLE, D. McHALE, D.M. DUNLOPand A. LAJTHA, Biochem. Biophys. Res. Comm. 141, 27-32 (1986). D.S. DUNLOPand A. NEIDLE, Anal. Biochem. 165, 38-44 (1987). D.W. ASWAD, Anal. Biochem. 138, 405-409 (1984). G. LEVI and G. MORISI, Brain Res. 26, 131-140 (1971). G.H. FISHER, N.M. GARCIA, I.L. PAYAN, R. CADILLA-PEREZRIOS, W.A. SHEREMATAand E.H. MAN, Biochem. Biophys. Res. Comm. 135, 683-687 (19861. E.H. MAN, G.H. FISHER, 1-L. PAYAN, R. CADILLA-PEREZRIOS, N.M. GARCIA, R. CHEMBURKAR, G. ARENDSand W.H. FREY II, J.Neurochem. 48, 510-515 (1987). R. SHAPIRA, K.D. WILKINSON and G. SHAPIRA, J.Neurochem. 49, 649-654 (1988). L.S. BRUNAUERand S. CLARKE, J. Biol. Chem. 261, 12538-12543 (1986). K. SUNIL and P. CHRISTEN, Eur. J. Biochem. 175, 433-438 (1988).