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Disturbances by galactose of the free amino acids of fetal rat brain
514
SHORT COMMUNICATIONS
BBA 23298
Disturbances by galactose of the free amino acids of fetal rat brain A number of studies have indicated that many hexoses inhibit amino acid uptake in kidney and intestine suggesting a relationship between the transport of certain sugars and amino acidsl, 2. Recent evidence indicates the possibility of a common carrier mechanism in the intestine for I>galactose and L-arginine 3. In view of the interference with cerebral function exerted by elevated blood galactose levels, this investigation was concerned with the effects of such elevated galactose levels in the maternal rat on the free amino acid pattern of the developing fetal brain. In addition, the opportunity was taken to investigate the possible biotransformations of galactose into amino acids of fetal rat brain. On the fourteenth day of pregnancy, Holtzman rats were placed on a diet consisting of 4° % galactose4; the controls received rat chow. On the twenty-first day of pregnancy, the fetuses from the control and galactose-fed animals were delivered by caesarean section, blotted with filter paper and frozen until the brains could be removed. Maternal blood was obtained by cardiac puncture, i h prior to sacrifice ,~/~C of D-Ii*C61galactose (spec. activity, 60 mC/mmole) was injected intraperitoneally into each of tile experimental and control groups. An additional group of animals was employed to test the effects of giving intraperitoneal injections of galactose to the pregnant rat. These animals were fed Purina chow throughout pregnancy and given 7oo mg I)-galactose/kg body weight in 2.o ml on the twenty-first day of pregnancy. They were sacrificed I h later and treated in the same manner as the animals fed galactose. Fetal brains from litter mates were pooled and considered one sample. The free amino acids were isolated 5 and quantitated 6. (ialactose was determined by gas chromatography 7. Radioactivity in individual amino acids was measured by passing the effluent from the amino acid analyzer through a Nuclear-Chicago Chromacell attached to a scintillation spectrometer. No gross anomalies were noted in any fetuses from maternal animals fed galactose. However, cataract formation in the fetal rat lens was similar to that reported by SIEGAL AND BERNSTEIN 4. N o cataracts were noted in the fetuses from animals injected with galactose. The feeding of galactose did not result in an elevation of blood galactose levels. This more than likely can be explained by the difficulty of choosing the proper time to sample the blood for elevated galactose. In contrast, those animals which received galactose by intraperitoneal injection had an increased blood level throughout the ensuing hour. These values ranged from 125 mg/Ioo ml of serum at 15 min to 225 nag/ IOO ml at 45 rain. Tile values for tile individual amino acids in maternal serum obtained on the twenty-first day of pregnancy indicate that the feeding of galactose had little effect on these compounds. This is no doubt the result of the difficulty of maintaining high blood galactose levels in the serum bv feeding alone. Also, with the exception of phenylalanine and tyrosine, no changes were observed in the concentrations of free amino acids in the fetal rat brains from galactose-fed maternal animals. On the other hand, when galactose was injected intraperitoneally, thus causing an immediate high Biochim. Biophys..4cfa, 13o (r066) 514-5 i6
SHORT COMMUNICATIONS TABLE FREE
515
1
AMINO
ACIDS
IN
FETAL
RAT
BRAIN
V a l u e s are e x p r e s s e d as t l m o l e s / g w e t tissue 4:._ S . E , and r e p r e s e n t t h e a v e r a g e of IO animals. _4micro
acid
Asparticacid Threonine Serine Glutamine Proline ( ; l u t a m i c acid Glvcine Alanine \'aline Methionine lsoleucine 1.eucine Tvrosine Phenvlalanine T - A m i n o b u t y r i c acid Ornithine l.vsine l-I'istidine Arginine
Control 1.64 o.67 1.14 3.IO 0.49 5-43 1.29 2.35 0.36 o.13 o.16 0.34 0.24 o.18 0.90 o.12 0.68 o.15 o.21
~ -~ ± ± ± ± ± i ± ± ± ~ ± ± ± ± ~ ~
o.o7 o.o 5 o.o9 o.17 0.08 0.37 0.07 o.15 0.02 o.oo8 o.oo6 0.02 0.02 o.oi 0.06 0.04 o.14 0.02 0.03
Exptl. feeding
Exptl. injection
1.46 ~ o . o 8 o.64 :: o.o 3 1.26 ± o . o 9 2 . 8 4 ± o. I7 0.46 ± 0.05 5.23 -- 0.27 1.42 ± 0 . 0 8 2.43 ± 0 . 2 0 0.36 L 0.03 o.16 ± o.oi o . 2 o .~ o.o2 o.41 :~ 0 . 0 4 o.3I* L 0.03 0.24*± 0.02 0.96 q 0.09 0 . 0 4 ~: 0 . 0 4 0-54 4:.- 0 . 0 6 o.13 ± 0.03 o.25 ± 0.03
t.65 ± o.i6 o.46* ~ o.o 5 o.89 ~: o . o 3 3.o9 ~ o.25 0 . 2 9 * * E 0.02 5.05 ± o.31 1.25 k 0.04 1.55"*± 0.06 0.23**± 0.02 o . o 8 " * ~ o.oo 7 o.io**± o.o2 0.22* ~ O.O1 o.21 ~ 0.03 o.19 ~ o.o2 0.82 ~ 0.02 0.04 -- 0 . 0 0 5 0.65 ± o.o 4 oat 4:.- 0 . 0 0 5 o.13"* ~ 0.006
* p < 0.05. ** P % o . o i .
blood galactose level, a number of changes were observed in the fetal brain amino acids (Table I). Threonine, proline, alanine, valine, methionine, isoleucine, leucine and arginine were significantly reduced in concentrations; the other amino acids were not affected. After the injection of uniformly labelled galactose, a number of amino acids in the fetal brain were radioactive at the end of I h. On the basis of percent distribution, glutamic acid showed the highest activity while alanine had approx. 12 O/o/ of the activity. The relative specific activity of the amino acids indicates that alanine, aspartic and glutamic acids were the most highly labelled, followed by 7-aminobutyric acid and glutamine. Maternal serum showed no radioactivity in the free amino acids. It is apparent from the data that the injection of large quantities of galactose with an ensuing elevated maternal blood level results in a disturbance in the free amino acid pool of the prenatal brain. The reasons for changes in concentrations of some amino acids and not in others, however, is not apparent. Presumably, transport mechanisms in the developing brain have been affected. Support for this concept is available from studies of galactose inhibition of amino acid transport into intestine and kidney. For example, fructose, glucose and galactose were the only sugars which inhibited amino acid uptake into kidney cortex slices 1. In the presence of galactose, inhibition of the transport of neutral amino acids into intestinal slices has been reported 8. More recently, evidence has been presented which suggests a common carrier mechanism for the transport of amino acids and sugars by the intestinO. The feeding of galactose to the pregnant rat caused only minimal changes in fetal brain amino acids and no changes in the flee amino acids of maternal serum. Biochim.
Biophys. Acta, 13o (1966) 5 1 4 - 5 1 6
516
SHORT COMMUNICATIONS
This no doubt can be related to the difficulty in maintaining elevated galactose blood levels b y feeding ad libitum, although others have been successful in doing so~. One of the problems, however, is that the rat, in contrast to patients with galactosemia, has the necessary enzyme systems for metabolizing galactose. Thus any galactose ingested is rapidly converted to glucose. Only by overloading the animal with massive doses of the sugar can any elevated blood level be attained for an appreciable length of time. The injection of uniformly labelled galactose I h before sacrificing the animals indicated that a rapid labelling of certain amino acids occurred in the fetal brain. It is not meant to imply that this conversion of galactose to the amino acids necessarily took place in the brain since there are m a n y tissues in both the maternal and fetal animal which could have affected such a conversion, although maternal plasma amino acids showed no radioactivity. Furthermore, the results of the entry of galactose carbon into amino acids is in agreement with those previously reported in adult rat braing, ~° with [14Clglucose. The results of the present study m a y have some application to the problem of galactosemia. It has been suggested that the cerebral disorder m a y begin in utero because of the transplacental passage of galactose 11. The nature of this disorder m a y be related to the distortion of the free amino acid pool of the developing brain. This could have serious consequences for protein synthesis 1~ and the production of important neuroregulators, hi the case of another inborn error of metabolism, phenylketonuria, it has already been demonstrated in adult and fetal rat brain that excessive circulating levels of phenylalanine causes a disturbance in the normal distribution of free amino acids~3, ~. This distortion in the amino acids m a y be a common factor in both conditions, This investigation was supported in part by National Institutes of Health Grants No. HDoo36I and HDoo37o from the National Institute of Child Health and H u m a n Development. Thanks are due to Dr. ,JOHN COPENHAVER for performing the blood galactose measurements by gas chromatography.
Nebraska Psychiatric Institute a~,d Department of Biochemistry, University of Nebraska College oJ Medicine, Omaha, Nebr. (U.S.A.) I 2 3 4 5 0 7 8 9
H. NE\VEY AND D. H. SMY'rH, A:at~re, 202 (1964) 400. S. THIER, M. FOX, L. ROSENBERC~ AND S. SE6AL, Biochim. Biophys. ~4cta, 93 (1904) to0. F. ALVARDO, Science, 15 (~960) IOlO. S. SEOAL AND H. BI~RNSTEIN, J . Pediat., 62 (~963) 363 . A. LAJTHA, S. FURST, A. GERSTI:,IN AND If. \VAELSCH, .]. Neurochem., i ( i 9 5 7 ) 2S9. D. H. SPACKMAN, W . H. STEIN AND S. MOORE, H n a l . Chem., 3o (1958) I~9o. J. 1:[. COPENHAVER, Hnal. Y3ioche>n., 17 (J900) 70" S. J. SAUNDERS AND K. J. [SSt~LBACHER, Biochim. tliophys. ~4cta, 1o2 (1965) 39,7. M. 1':. (~AI'I'ONDE, l). 1{. DAHL AND 1"2. A. ('. ELL1OTT, Biochem. J., 94 (1965) 345-
xo J. E. CREM~'~a,.l" N e u r o c h e n T . , ~1 12 13 14
MICHAEL J. CARVER
TI
(J904) 165.
M. SI, AXz AND S. SE6AL, J. Pedial., 67 (1905) 438. S. J~OBERTS aXl:~ B. S. MORELOS, J. Ncurochem., 12 (~905) 373M. J. CARVl'ZR, .[. Ne**rochem., z2 (I965) 45M. J. CARVV:R, J. H. COPENHAVER AND R. A. SERPAN, J. Neurochem., t2 (t905) 857.
Received .June 28th, I966 Revised manuscript received September I2th, 1966 Biochim. 13iophys. ~4cla, 13o (19o0) 514 51o
SHORT COMMUNICATIONS
BBA 23298
Disturbances by galactose of the free amino acids of fetal rat brain A number of studies have indicated that many hexoses inhibit amino acid uptake in kidney and intestine suggesting a relationship between the transport of certain sugars and amino acidsl, 2. Recent evidence indicates the possibility of a common carrier mechanism in the intestine for I>galactose and L-arginine 3. In view of the interference with cerebral function exerted by elevated blood galactose levels, this investigation was concerned with the effects of such elevated galactose levels in the maternal rat on the free amino acid pattern of the developing fetal brain. In addition, the opportunity was taken to investigate the possible biotransformations of galactose into amino acids of fetal rat brain. On the fourteenth day of pregnancy, Holtzman rats were placed on a diet consisting of 4° % galactose4; the controls received rat chow. On the twenty-first day of pregnancy, the fetuses from the control and galactose-fed animals were delivered by caesarean section, blotted with filter paper and frozen until the brains could be removed. Maternal blood was obtained by cardiac puncture, i h prior to sacrifice ,~/~C of D-Ii*C61galactose (spec. activity, 60 mC/mmole) was injected intraperitoneally into each of tile experimental and control groups. An additional group of animals was employed to test the effects of giving intraperitoneal injections of galactose to the pregnant rat. These animals were fed Purina chow throughout pregnancy and given 7oo mg I)-galactose/kg body weight in 2.o ml on the twenty-first day of pregnancy. They were sacrificed I h later and treated in the same manner as the animals fed galactose. Fetal brains from litter mates were pooled and considered one sample. The free amino acids were isolated 5 and quantitated 6. (ialactose was determined by gas chromatography 7. Radioactivity in individual amino acids was measured by passing the effluent from the amino acid analyzer through a Nuclear-Chicago Chromacell attached to a scintillation spectrometer. No gross anomalies were noted in any fetuses from maternal animals fed galactose. However, cataract formation in the fetal rat lens was similar to that reported by SIEGAL AND BERNSTEIN 4. N o cataracts were noted in the fetuses from animals injected with galactose. The feeding of galactose did not result in an elevation of blood galactose levels. This more than likely can be explained by the difficulty of choosing the proper time to sample the blood for elevated galactose. In contrast, those animals which received galactose by intraperitoneal injection had an increased blood level throughout the ensuing hour. These values ranged from 125 mg/Ioo ml of serum at 15 min to 225 nag/ IOO ml at 45 rain. Tile values for tile individual amino acids in maternal serum obtained on the twenty-first day of pregnancy indicate that the feeding of galactose had little effect on these compounds. This is no doubt the result of the difficulty of maintaining high blood galactose levels in the serum bv feeding alone. Also, with the exception of phenylalanine and tyrosine, no changes were observed in the concentrations of free amino acids in the fetal rat brains from galactose-fed maternal animals. On the other hand, when galactose was injected intraperitoneally, thus causing an immediate high Biochim. Biophys..4cfa, 13o (r066) 514-5 i6
SHORT COMMUNICATIONS TABLE FREE
515
1
AMINO
ACIDS
IN
FETAL
RAT
BRAIN
V a l u e s are e x p r e s s e d as t l m o l e s / g w e t tissue 4:._ S . E , and r e p r e s e n t t h e a v e r a g e of IO animals. _4micro
acid
Asparticacid Threonine Serine Glutamine Proline ( ; l u t a m i c acid Glvcine Alanine \'aline Methionine lsoleucine 1.eucine Tvrosine Phenvlalanine T - A m i n o b u t y r i c acid Ornithine l.vsine l-I'istidine Arginine
Control 1.64 o.67 1.14 3.IO 0.49 5-43 1.29 2.35 0.36 o.13 o.16 0.34 0.24 o.18 0.90 o.12 0.68 o.15 o.21
~ -~ ± ± ± ± ± i ± ± ± ~ ± ± ± ± ~ ~
o.o7 o.o 5 o.o9 o.17 0.08 0.37 0.07 o.15 0.02 o.oo8 o.oo6 0.02 0.02 o.oi 0.06 0.04 o.14 0.02 0.03
Exptl. feeding
Exptl. injection
1.46 ~ o . o 8 o.64 :: o.o 3 1.26 ± o . o 9 2 . 8 4 ± o. I7 0.46 ± 0.05 5.23 -- 0.27 1.42 ± 0 . 0 8 2.43 ± 0 . 2 0 0.36 L 0.03 o.16 ± o.oi o . 2 o .~ o.o2 o.41 :~ 0 . 0 4 o.3I* L 0.03 0.24*± 0.02 0.96 q 0.09 0 . 0 4 ~: 0 . 0 4 0-54 4:.- 0 . 0 6 o.13 ± 0.03 o.25 ± 0.03
t.65 ± o.i6 o.46* ~ o.o 5 o.89 ~: o . o 3 3.o9 ~ o.25 0 . 2 9 * * E 0.02 5.05 ± o.31 1.25 k 0.04 1.55"*± 0.06 0.23**± 0.02 o . o 8 " * ~ o.oo 7 o.io**± o.o2 0.22* ~ O.O1 o.21 ~ 0.03 o.19 ~ o.o2 0.82 ~ 0.02 0.04 -- 0 . 0 0 5 0.65 ± o.o 4 oat 4:.- 0 . 0 0 5 o.13"* ~ 0.006
* p < 0.05. ** P % o . o i .
blood galactose level, a number of changes were observed in the fetal brain amino acids (Table I). Threonine, proline, alanine, valine, methionine, isoleucine, leucine and arginine were significantly reduced in concentrations; the other amino acids were not affected. After the injection of uniformly labelled galactose, a number of amino acids in the fetal brain were radioactive at the end of I h. On the basis of percent distribution, glutamic acid showed the highest activity while alanine had approx. 12 O/o/ of the activity. The relative specific activity of the amino acids indicates that alanine, aspartic and glutamic acids were the most highly labelled, followed by 7-aminobutyric acid and glutamine. Maternal serum showed no radioactivity in the free amino acids. It is apparent from the data that the injection of large quantities of galactose with an ensuing elevated maternal blood level results in a disturbance in the free amino acid pool of the prenatal brain. The reasons for changes in concentrations of some amino acids and not in others, however, is not apparent. Presumably, transport mechanisms in the developing brain have been affected. Support for this concept is available from studies of galactose inhibition of amino acid transport into intestine and kidney. For example, fructose, glucose and galactose were the only sugars which inhibited amino acid uptake into kidney cortex slices 1. In the presence of galactose, inhibition of the transport of neutral amino acids into intestinal slices has been reported 8. More recently, evidence has been presented which suggests a common carrier mechanism for the transport of amino acids and sugars by the intestinO. The feeding of galactose to the pregnant rat caused only minimal changes in fetal brain amino acids and no changes in the flee amino acids of maternal serum. Biochim.
Biophys. Acta, 13o (1966) 5 1 4 - 5 1 6
516
SHORT COMMUNICATIONS
This no doubt can be related to the difficulty in maintaining elevated galactose blood levels b y feeding ad libitum, although others have been successful in doing so~. One of the problems, however, is that the rat, in contrast to patients with galactosemia, has the necessary enzyme systems for metabolizing galactose. Thus any galactose ingested is rapidly converted to glucose. Only by overloading the animal with massive doses of the sugar can any elevated blood level be attained for an appreciable length of time. The injection of uniformly labelled galactose I h before sacrificing the animals indicated that a rapid labelling of certain amino acids occurred in the fetal brain. It is not meant to imply that this conversion of galactose to the amino acids necessarily took place in the brain since there are m a n y tissues in both the maternal and fetal animal which could have affected such a conversion, although maternal plasma amino acids showed no radioactivity. Furthermore, the results of the entry of galactose carbon into amino acids is in agreement with those previously reported in adult rat braing, ~° with [14Clglucose. The results of the present study m a y have some application to the problem of galactosemia. It has been suggested that the cerebral disorder m a y begin in utero because of the transplacental passage of galactose 11. The nature of this disorder m a y be related to the distortion of the free amino acid pool of the developing brain. This could have serious consequences for protein synthesis 1~ and the production of important neuroregulators, hi the case of another inborn error of metabolism, phenylketonuria, it has already been demonstrated in adult and fetal rat brain that excessive circulating levels of phenylalanine causes a disturbance in the normal distribution of free amino acids~3, ~. This distortion in the amino acids m a y be a common factor in both conditions, This investigation was supported in part by National Institutes of Health Grants No. HDoo36I and HDoo37o from the National Institute of Child Health and H u m a n Development. Thanks are due to Dr. ,JOHN COPENHAVER for performing the blood galactose measurements by gas chromatography.
Nebraska Psychiatric Institute a~,d Department of Biochemistry, University of Nebraska College oJ Medicine, Omaha, Nebr. (U.S.A.) I 2 3 4 5 0 7 8 9
H. NE\VEY AND D. H. SMY'rH, A:at~re, 202 (1964) 400. S. THIER, M. FOX, L. ROSENBERC~ AND S. SE6AL, Biochim. Biophys. ~4cta, 93 (1904) to0. F. ALVARDO, Science, 15 (~960) IOlO. S. SEOAL AND H. BI~RNSTEIN, J . Pediat., 62 (~963) 363 . A. LAJTHA, S. FURST, A. GERSTI:,IN AND If. \VAELSCH, .]. Neurochem., i ( i 9 5 7 ) 2S9. D. H. SPACKMAN, W . H. STEIN AND S. MOORE, H n a l . Chem., 3o (1958) I~9o. J. 1:[. COPENHAVER, Hnal. Y3ioche>n., 17 (J900) 70" S. J. SAUNDERS AND K. J. [SSt~LBACHER, Biochim. tliophys. ~4cta, 1o2 (1965) 39,7. M. 1':. (~AI'I'ONDE, l). 1{. DAHL AND 1"2. A. ('. ELL1OTT, Biochem. J., 94 (1965) 345-
xo J. E. CREM~'~a,.l" N e u r o c h e n T . , ~1 12 13 14
MICHAEL J. CARVER
TI
(J904) 165.
M. SI, AXz AND S. SE6AL, J. Pedial., 67 (1905) 438. S. J~OBERTS aXl:~ B. S. MORELOS, J. Ncurochem., 12 (~905) 373M. J. CARVl'ZR, .[. Ne**rochem., z2 (I965) 45M. J. CARVV:R, J. H. COPENHAVER AND R. A. SERPAN, J. Neurochem., t2 (t905) 857.
Received .June 28th, I966 Revised manuscript received September I2th, 1966 Biochim. 13iophys. ~4cla, 13o (19o0) 514 51o