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Postnatal changes in the concentration of carnitine and acylcarnitines in rat brain
Developmental Brain Research, 8 (1983) 381-384 Elsevier
381
Postnatal changes in the concentration of carnitine and acylcarnitines in rat brain ANTHONY J. MORRIS and ERIC M. CAREY Department of Biochemistry, The University, Sheffield SIO 2TN (U. K.) (Accepted February 1st, 1983) Key words: carnitine, total, free - - short-chain, long chain - - acylcarnitines, concentration-development - - rat brain
The concentration of total and free carnitine (TC and FC) together with short-chain and long-chain acylcarnitines (SCAC and LCAC) has been determined in whole brain of female rats from birth to maturity. The concentration of TC and SCAC increases postnatally to reach a peak 10 days after birth. The concentration of LCAC is high relative to adult brain between 1 and 10 days of age before falling to a low level. The change in LCAC concentration correlates with previously described developmental changes of palmitoyl-CoA: carnitine acyl transferase activity and fatty acid oxidation. The ratio of LCAC/FC declines postnatally while the SCAC/FC ratio is high at birth and increases further in adult brain. The results are consistent with the concepts of carnitine participating in exchange of short-chain acyl groups and in the transfer of fatty acids into mitochondria as an alternative energy supply in neonatal rat brain. The function of carnitine in the transport of longchain fatty acids across the mitochondrial membrane is well established for a n u m b e r of tissues such as liver, skeletal and cardiac muscle and brown adipose tissue which are known to oxidize fatty acid as a major source of energy. Carnitine promotes both fatty acid oxidation and ketogenesis in the liver, giving rise to a supply of ketones for oxidation by extrahepatic tissues 13which is of major importance in the neonate receiving a high fat milk diet 5. Carnitine also promotes the oxidation of fatty acid in brown adipose tissue during non-shivering thermogenesis7, 8. Although carnitine and acylcarnitines are present in nervous tissues 6 and the carnitine-mediated translocation of long-chain fatty acid across the inner mitochondrial m e m b r a n e occurs with mitochondria isolated from the brain as with other tissues 1, little is known about the importance of carnitine in the development of the brain. Postnatally the brain depends to a considerable extent on oxidizable substrates other than glucose 9. The plasma concentration of both ketone bodies and lipids are elevated postnatally in the rat, while at the same time there is a postnatal increase in the activity of enzymes in the pathway for ketone utilization9. Fatty acids are also oxidized to a greater extent by the neonatal brain in vivo 17, by tissue homogenates 0165-3806/83/$03.00 (~) 1983 Elsevier Science Publishers B.V.
and isolated mitochondria compared with the adult brain 1. Fatty acid oxidation shows highest activity 7-10 days after birth, coincident with an elevated activity of carnitine acyl transferase in the developing rat brain 18. The concentration of carnitine in adult brain tissue is low compared with tissues known to oxidise fatty acids in vivo such as skeletal and cardiac muscle n, and there is a good correlation between the capacity for fatty acid oxidation and the tissue carnitine concentration 2. There is little information on the concentration of carnitine and its esters in the brain tissue of the neonate, at a time when the brain may obtain a significant proportion of its energy requirements from fatty acid oxidation. In this report, we describe the postnatal changes in the concentration of total and free carnitine and also short- and long-chain acylcarnitines in developing rat brain. All the animals used were female Wistar rats of an inbred albino strain from the Sheffield University animal house colony. Animals were maintained on a 12 h lighting schedule at a constant temperature (22 °C) and humidity (45%). Animals were permitted free access to food and water. The stock diet was the C.R.M. diet supplied by Labsure Animal Foods (Christopher Hill Group, Poole, Dorset). The composition of the diet was 2.35% fat, 17.45% protein,
382 73.65% carbohydrate, 4.26% fiber and 2.5% salts. The day on which a litter was recorded was counted as day 0 as the animals normally gave birth at night. Litters contained 9-14 pups and only the females were used for the estimation of brain carnitine concentration. Animals were weaned at 21 days onto the stock diet. Animals were decapitated without anesthesia and the whole brain, including cerebellum and brainstem, was removed within 10 s, weighed and immediately homogenized in ice-cold 0.3 M perchloric acid at a ratio of 1 ml perchloric acid/g wet weight o f tissue 14. The homogenate was centrifuged at 3000 g for 10 min and at 4 °C. Free carnitine and short-chain acylcarnitine remain in the supernatant and longchain acylcarnitine is precipitated in perchloric acid. Free carnitine was assayed in 100/d of the neutralized perchloric acid extract. Carnitine was released from short-chain acylcarnitines by alkaline hydrolysis 14and the carnitine in the hydrolyzed extract minus the free carnitine gives the short-chain acylcarnitine. After washing the perchloric acid precipitate with distilled water, long-chain acylcarnitines were hydrolyzed in alkaline solution and neutralized with perchloric acid. The released carnitine was determined in 100 ¢tl of the extract. The total tissue carnitine concentration is the sum of the free plus short-chain acylcarnitine and the long-chain acylcarnitine. Carnitine was assayed using the radioisotopic method of McGarry and Fosterl2, with minor modifications. The assay contained (in 1.0 ml vol.): 100 ~mol Tris-HCl, pH 7.3, 2j~mol sodium tetrathionate, 110 nmol [1-14C]-acetyl-CoA (spec. act. 0.23 Ci/moi) and 100 BI of neutralized tissue extract. A standard DL-carnitine solution was freshly prepared prior to carrying out each set of assays, and a carnitine standard curve (5,10 and 40 nmol l,-carnitine) and carnitine blanks were included in each set of assays. Standards were assayed in triplicate and the assay of carnitine in tissue extracts was performed in duplicate. With standard and blank assays, 100¢tl of a synthetic tissue extract (prepared by neutralizing 0.3 M perchloric acid, as carried out on deproteinized tissue extracts) was added to each assay. Reactions were begun by the addition of 5 ~1 carnitine acetyltransferase (1 unit) and allowed to stand at room temperature for 30 min. Then 0.3 ml of a stirred slurry of Dowex 2-X8 (27% by weight of dry resin)
was added, agitated with a vortex mixer and allowed to stand on ice for 10 rain. This was repeated twice, followed by transfer of 0.5 ml of the supernatant containing [14C]acetyl-carnitine to 10 ml Triton-toluene (1:2, v/v) containing 0.4% 2,5-diphenyloxazole and 0.02% 1,4-di-[2-(5-phenyloxazolyl)] benzene. Samples were left overnight at room temperature prior to quantitation of radioactivity using a Philips PW4700 liquid scintillation counter. The assay was found to be linear with up to 400 nmol L-carnitine in the assay, and the cpm/nmol Lcarnitine (129 _+ 4.4 (S.D.)) was calculated from the carnitine standards. This was applied to the determination of the amount of carnitine in the tissue extracts. The external standard ratio obtained with the standards and tissue extracts was constant and quench correction was unnecessary. The difference between duplicates was routinely less than 3% of the mean value. The concentration of carnitine (free plus esterifled), free carnitine, short-chain acylcarnitine and long-chain acylcarnitine in brain tissue of the female rat is shown in Fig. 1. The total carnitine, short-chain acylcarnitine and free carnitine concentrations increased from 1 day postnatal age to peak at 10 days of age, followed by a decline. The concentration of total carnitine and short-chain acylcarnitine showed a slight increase between 21 days of age (weaning) and 96 days of age. The concentration of free carnitine fell progressively from the peak at t0 days, The concentration of long-chain acylcarnitine was higher between 1 and 10 days postnatal age. At 5 days the concentration was marginally higher than one day after birth (P < 0.5). There was a significant fall in the concentration of long-chain acylcarnitine from 10 days of age through to the adult stage. The ratio of the concentrations of long-chain acylcarnitine to free carnitine decreased postnatally, with the highest value at I day postnatal age, while the ratio of short-chain acylcarnitine to free carnitine showed a slight fall between 10 and 20 days of age, with the highest ratio occurring at the adult stage, where the concentration of free carnitine was at its lowest. A large proportion of carnitine is esterified to short-chain acyl groups, consistent with the carnitine mediated translocation of acetyl units between the mitochondrion and cytoplasm 6. The ratio of shortchain acylcarnitine to free carnitine is very high in
383
brain tissue c o m p a r e d with o t h e r tissues. In the newborn rat, milk is the m a j o r source of carnitine, while the synthesis of carnitine from 7-butyrobetaine by the liver, is low in the foetus but increases to reach adult values by the eighth postnatal day 8. In the neonatal period, carnitine is accumulated in those tissues which oxidize fatty acidsS. I m m e d i a t e l y after birth the uptake into brown adipose tissue exceeds that of the liver, heart and skeletal muscle. The postnatal accumulation of carnitine by the developing nervous system o b s e r v e d here occurs subsequent to the increase in plasma carnitine ~6 and the ac-
cumulation in brown adipose tissue. T h e timing of the increase in the carnitine concentration in the brain coincides with adaptive changes p r o m o t i n g fatty acid uptake 4 and metabolism18. In an earlier study, Carroll and Elkin 3 c o m p a r e d the concentration of total carnitine and short-chain acylcarnitine in rat brain at ages of less than 5 days p o s t p a r t u m with adult brain. They found that the total carnitine concentration was higher in adult brain c o m p a r e d with neonatal brain, but they did not determine the carnitine concentration t h r o u g h o u t the period of brain development. The higher concentration of long-chain fatty acylcarnitine in the neonatal brain b e t w e e n 1 and 10 days of age is consistent with an increased translocation of fatty acids across the inner mitochondrial m e m b r a n e at this time. The decline in the concentration of longchain acylcarnitines b e y o n d 10 days of age and the fall in the long-chain acylcarnitine to carnitine ratio suggests that fatty acid oxidation m a y b e c o m e less significant in the supply of energy for the adult brain. T h e r e were significant differences b e t w e e n litters in the concentration of total and short-chain acylcarnitine in the brain from birth to 15 days of age. Beyond 15 days there was no significant difference between litters. The difference was most p r o n o u n c e d at birth. T h e r e was no significant difference b e t w e e n female and male rats in the carnitine content of the brain at 10 and 20 days of age (results not p r e s e n t e d ) . No comparison was m a d e at other ages. In skeletal muscle, the carnitine content of the male is higher than the female at maturity but not during the weaning period 2. Low levels of serum camitine as seen in systemic carnitine deficiency result in pathological changes in the human infant brain including e n c e p h a l o p a t h y and the accumulation of neutral lipid 10. A deficiency of serum carnitine at a critical p e r i o d of brain develo p m e n t also occurs in p r e m a t u r e infants receiving total p a r e n t e r a l nutrition 15. It is not known what level of carnitine is r e q u i r e d for n o r m a l brain development.
1 Beattie, D. and Basford, J., Brain mitochondria III fatty acid oxidation by bovine brain mitochondria, J. Neurochem., 12 (1965) 103-111. 2 Borum, P. R., Variation in tissue carnitine concentrations with age and sex in the rat, Biochem. J., 176 (1978) 677-681.
3 Carroll, J. E. and Elkin, D. J., A possible role for carnitine in neonatal brain, Ann. Neurol., 6 (1979) 183-184. 4 Chajek, T., Stein, O. and Stein, Y., Pre- and postnatal development of iipoprotein lipase and hepatic triglyceride hydrolase activity in rat tissues., Atherosclerosis, 26 (1977) 549-561.
800
xx, E
400"
g 0 '0
100
Post-natal Age (days)
Fig. 1. Postnatal changes in the concentration of total carnitine, free carnitine, short-chain acylcarnitine and long-chain acylcarnitine in female rat brain. The concentration of carnitine is expressed as nmol/g wet weight of tissue. 0 , total carnitine; &, short-chain acylcarnitine; A, long-chain acylcarnitine; O, free carnitine. The bars shown for total and short-chain acylcarnitine indicate the value of the S.E.M. The S.E.M. values for long-chain acylcarnitine and free carnitine are too small to be included. The values for the S.E.M. varied from 6 to 24% of the mean value. The level of significance of values at different ages compared with the 1-day values were determined using Students' t-test. For total carnitine and short-chain acylcarnitine this is indicated as X (P < 0.05), XX (P < 0.01) and XXX (P < 0.001). For long-chain acylcarnitine and free carnitine the level of significance at 1,5 and 10 days compared with the values at 96 days was P < 0.025. The number of tissue samples, animals and litters used at each age was: 1 day, 5 tissue samples (combining brains of two animals from the same litter for each sample) from 4 litters; 5 days, 9 tissue samples (two brains for each sample) from 4 litters; 10 days, 16 animals from 8 litters; 15 days, 6 animals from 3 litters; 20 days, 19 animals from 6 litters; 30 days, 6 animals from 6 litters; 96 days, 3 animals from 3 litters.
384 5 Foster, P. C. and Bailey, E., Changes in hepatic fatty acid degradation and blood lipid and ketone body content during development of the rat, Enzyme, 21 (1976) 397-407. 6 Fritz, I. B., Carnitine and its role in fatty acid metabolism, Advanc. Lipid Res., 1 (1963) 285-336. 7 Hahn, P. and Skala, J. P., The role of carnitine in brown adipose tissue of suckling rats, Comp. Biochem. Physiol.. 51B (1975) 507-515. 8 Hahn, P., Secombe, D. W. and Towell, M. E., The perinatal role of carnitine. In M. Monset-Couchard and A. Mintowski (Eds.), Physiological and Biochemical basis for Perinatal Medicine, Karger, Basel, 1981, pp. 187-198. 9 Hawkins, R. A., Williamson, D. H. and Krebs, H. A., Ketone-body utilization by adult and suckling rat brain in vivo, Biochem. J., 122 (1971) 13-18. 10 Karpata, G., Carpenter, S., Engel, A., Waiters, G., Allen, J., Rothman, S., Klassen, G. and Mamer, O. A., The syndrome of systemic carnitine deficiency - - clinical morphological and pathophysiological features, Neurology, 25 (1975) 16-24. 11 Marquis, N. R. and Fritz, I. B., The distribution of carnitine, acetylcarnitine and carnitine acetyltransferase in rat tissues, J. biol. Chem., 240 (1965) 2193-2196.
12 McGarry, J. D. and Foster, D. W., An improved and sire plified radioisotope assay for the determination of free and esterified carnitine, J, lipid Res., 17 (1976) 277--281. 13 McGarry, J. D, and Foster, D. W., Regulati~m o! hepatic fatty acid oxidation and ketone body production, Ann. Rev. Biochem., 49 (1980) 395-420. 14 Pace, J. A., Wannemacher, R. W. and Neufeld, tt, A., Improved radiochemical assay for carnitine and its derivatives in plasma and tissue extracts, Clin. Chem. 2nd, {1978) 32-35. 15 Penn, D., Carnitine deficiency in premature infants receiving total parenteral nutrition., Early' Human Develop., 4 (1980) 23-34. 16 Seccombe, D. W., Hahn, P. and Novak. M.. The effect of diet and development on blood levels of free and esterified carnitine in the rat, Biochim. biophys. Acta. 528 (1978) 483-489. 17 Spitzer, J. J., Application of tracers in studying free fatty acid metabolism of various organs in vivo, Fed. Proc., 34 (1975) 2242-2245. 18 Warshaw, J. B. and Terry, M. L., Cellular energy metabolism during fetal development. VI Fatty acid oxidation by developing brain, Develop. biol., 52 (1976) 161-166.
381
Postnatal changes in the concentration of carnitine and acylcarnitines in rat brain ANTHONY J. MORRIS and ERIC M. CAREY Department of Biochemistry, The University, Sheffield SIO 2TN (U. K.) (Accepted February 1st, 1983) Key words: carnitine, total, free - - short-chain, long chain - - acylcarnitines, concentration-development - - rat brain
The concentration of total and free carnitine (TC and FC) together with short-chain and long-chain acylcarnitines (SCAC and LCAC) has been determined in whole brain of female rats from birth to maturity. The concentration of TC and SCAC increases postnatally to reach a peak 10 days after birth. The concentration of LCAC is high relative to adult brain between 1 and 10 days of age before falling to a low level. The change in LCAC concentration correlates with previously described developmental changes of palmitoyl-CoA: carnitine acyl transferase activity and fatty acid oxidation. The ratio of LCAC/FC declines postnatally while the SCAC/FC ratio is high at birth and increases further in adult brain. The results are consistent with the concepts of carnitine participating in exchange of short-chain acyl groups and in the transfer of fatty acids into mitochondria as an alternative energy supply in neonatal rat brain. The function of carnitine in the transport of longchain fatty acids across the mitochondrial membrane is well established for a n u m b e r of tissues such as liver, skeletal and cardiac muscle and brown adipose tissue which are known to oxidize fatty acid as a major source of energy. Carnitine promotes both fatty acid oxidation and ketogenesis in the liver, giving rise to a supply of ketones for oxidation by extrahepatic tissues 13which is of major importance in the neonate receiving a high fat milk diet 5. Carnitine also promotes the oxidation of fatty acid in brown adipose tissue during non-shivering thermogenesis7, 8. Although carnitine and acylcarnitines are present in nervous tissues 6 and the carnitine-mediated translocation of long-chain fatty acid across the inner mitochondrial m e m b r a n e occurs with mitochondria isolated from the brain as with other tissues 1, little is known about the importance of carnitine in the development of the brain. Postnatally the brain depends to a considerable extent on oxidizable substrates other than glucose 9. The plasma concentration of both ketone bodies and lipids are elevated postnatally in the rat, while at the same time there is a postnatal increase in the activity of enzymes in the pathway for ketone utilization9. Fatty acids are also oxidized to a greater extent by the neonatal brain in vivo 17, by tissue homogenates 0165-3806/83/$03.00 (~) 1983 Elsevier Science Publishers B.V.
and isolated mitochondria compared with the adult brain 1. Fatty acid oxidation shows highest activity 7-10 days after birth, coincident with an elevated activity of carnitine acyl transferase in the developing rat brain 18. The concentration of carnitine in adult brain tissue is low compared with tissues known to oxidise fatty acids in vivo such as skeletal and cardiac muscle n, and there is a good correlation between the capacity for fatty acid oxidation and the tissue carnitine concentration 2. There is little information on the concentration of carnitine and its esters in the brain tissue of the neonate, at a time when the brain may obtain a significant proportion of its energy requirements from fatty acid oxidation. In this report, we describe the postnatal changes in the concentration of total and free carnitine and also short- and long-chain acylcarnitines in developing rat brain. All the animals used were female Wistar rats of an inbred albino strain from the Sheffield University animal house colony. Animals were maintained on a 12 h lighting schedule at a constant temperature (22 °C) and humidity (45%). Animals were permitted free access to food and water. The stock diet was the C.R.M. diet supplied by Labsure Animal Foods (Christopher Hill Group, Poole, Dorset). The composition of the diet was 2.35% fat, 17.45% protein,
382 73.65% carbohydrate, 4.26% fiber and 2.5% salts. The day on which a litter was recorded was counted as day 0 as the animals normally gave birth at night. Litters contained 9-14 pups and only the females were used for the estimation of brain carnitine concentration. Animals were weaned at 21 days onto the stock diet. Animals were decapitated without anesthesia and the whole brain, including cerebellum and brainstem, was removed within 10 s, weighed and immediately homogenized in ice-cold 0.3 M perchloric acid at a ratio of 1 ml perchloric acid/g wet weight o f tissue 14. The homogenate was centrifuged at 3000 g for 10 min and at 4 °C. Free carnitine and short-chain acylcarnitine remain in the supernatant and longchain acylcarnitine is precipitated in perchloric acid. Free carnitine was assayed in 100/d of the neutralized perchloric acid extract. Carnitine was released from short-chain acylcarnitines by alkaline hydrolysis 14and the carnitine in the hydrolyzed extract minus the free carnitine gives the short-chain acylcarnitine. After washing the perchloric acid precipitate with distilled water, long-chain acylcarnitines were hydrolyzed in alkaline solution and neutralized with perchloric acid. The released carnitine was determined in 100 ¢tl of the extract. The total tissue carnitine concentration is the sum of the free plus short-chain acylcarnitine and the long-chain acylcarnitine. Carnitine was assayed using the radioisotopic method of McGarry and Fosterl2, with minor modifications. The assay contained (in 1.0 ml vol.): 100 ~mol Tris-HCl, pH 7.3, 2j~mol sodium tetrathionate, 110 nmol [1-14C]-acetyl-CoA (spec. act. 0.23 Ci/moi) and 100 BI of neutralized tissue extract. A standard DL-carnitine solution was freshly prepared prior to carrying out each set of assays, and a carnitine standard curve (5,10 and 40 nmol l,-carnitine) and carnitine blanks were included in each set of assays. Standards were assayed in triplicate and the assay of carnitine in tissue extracts was performed in duplicate. With standard and blank assays, 100¢tl of a synthetic tissue extract (prepared by neutralizing 0.3 M perchloric acid, as carried out on deproteinized tissue extracts) was added to each assay. Reactions were begun by the addition of 5 ~1 carnitine acetyltransferase (1 unit) and allowed to stand at room temperature for 30 min. Then 0.3 ml of a stirred slurry of Dowex 2-X8 (27% by weight of dry resin)
was added, agitated with a vortex mixer and allowed to stand on ice for 10 rain. This was repeated twice, followed by transfer of 0.5 ml of the supernatant containing [14C]acetyl-carnitine to 10 ml Triton-toluene (1:2, v/v) containing 0.4% 2,5-diphenyloxazole and 0.02% 1,4-di-[2-(5-phenyloxazolyl)] benzene. Samples were left overnight at room temperature prior to quantitation of radioactivity using a Philips PW4700 liquid scintillation counter. The assay was found to be linear with up to 400 nmol L-carnitine in the assay, and the cpm/nmol Lcarnitine (129 _+ 4.4 (S.D.)) was calculated from the carnitine standards. This was applied to the determination of the amount of carnitine in the tissue extracts. The external standard ratio obtained with the standards and tissue extracts was constant and quench correction was unnecessary. The difference between duplicates was routinely less than 3% of the mean value. The concentration of carnitine (free plus esterifled), free carnitine, short-chain acylcarnitine and long-chain acylcarnitine in brain tissue of the female rat is shown in Fig. 1. The total carnitine, short-chain acylcarnitine and free carnitine concentrations increased from 1 day postnatal age to peak at 10 days of age, followed by a decline. The concentration of total carnitine and short-chain acylcarnitine showed a slight increase between 21 days of age (weaning) and 96 days of age. The concentration of free carnitine fell progressively from the peak at t0 days, The concentration of long-chain acylcarnitine was higher between 1 and 10 days postnatal age. At 5 days the concentration was marginally higher than one day after birth (P < 0.5). There was a significant fall in the concentration of long-chain acylcarnitine from 10 days of age through to the adult stage. The ratio of the concentrations of long-chain acylcarnitine to free carnitine decreased postnatally, with the highest value at I day postnatal age, while the ratio of short-chain acylcarnitine to free carnitine showed a slight fall between 10 and 20 days of age, with the highest ratio occurring at the adult stage, where the concentration of free carnitine was at its lowest. A large proportion of carnitine is esterified to short-chain acyl groups, consistent with the carnitine mediated translocation of acetyl units between the mitochondrion and cytoplasm 6. The ratio of shortchain acylcarnitine to free carnitine is very high in
383
brain tissue c o m p a r e d with o t h e r tissues. In the newborn rat, milk is the m a j o r source of carnitine, while the synthesis of carnitine from 7-butyrobetaine by the liver, is low in the foetus but increases to reach adult values by the eighth postnatal day 8. In the neonatal period, carnitine is accumulated in those tissues which oxidize fatty acidsS. I m m e d i a t e l y after birth the uptake into brown adipose tissue exceeds that of the liver, heart and skeletal muscle. The postnatal accumulation of carnitine by the developing nervous system o b s e r v e d here occurs subsequent to the increase in plasma carnitine ~6 and the ac-
cumulation in brown adipose tissue. T h e timing of the increase in the carnitine concentration in the brain coincides with adaptive changes p r o m o t i n g fatty acid uptake 4 and metabolism18. In an earlier study, Carroll and Elkin 3 c o m p a r e d the concentration of total carnitine and short-chain acylcarnitine in rat brain at ages of less than 5 days p o s t p a r t u m with adult brain. They found that the total carnitine concentration was higher in adult brain c o m p a r e d with neonatal brain, but they did not determine the carnitine concentration t h r o u g h o u t the period of brain development. The higher concentration of long-chain fatty acylcarnitine in the neonatal brain b e t w e e n 1 and 10 days of age is consistent with an increased translocation of fatty acids across the inner mitochondrial m e m b r a n e at this time. The decline in the concentration of longchain acylcarnitines b e y o n d 10 days of age and the fall in the long-chain acylcarnitine to carnitine ratio suggests that fatty acid oxidation m a y b e c o m e less significant in the supply of energy for the adult brain. T h e r e were significant differences b e t w e e n litters in the concentration of total and short-chain acylcarnitine in the brain from birth to 15 days of age. Beyond 15 days there was no significant difference between litters. The difference was most p r o n o u n c e d at birth. T h e r e was no significant difference b e t w e e n female and male rats in the carnitine content of the brain at 10 and 20 days of age (results not p r e s e n t e d ) . No comparison was m a d e at other ages. In skeletal muscle, the carnitine content of the male is higher than the female at maturity but not during the weaning period 2. Low levels of serum camitine as seen in systemic carnitine deficiency result in pathological changes in the human infant brain including e n c e p h a l o p a t h y and the accumulation of neutral lipid 10. A deficiency of serum carnitine at a critical p e r i o d of brain develo p m e n t also occurs in p r e m a t u r e infants receiving total p a r e n t e r a l nutrition 15. It is not known what level of carnitine is r e q u i r e d for n o r m a l brain development.
1 Beattie, D. and Basford, J., Brain mitochondria III fatty acid oxidation by bovine brain mitochondria, J. Neurochem., 12 (1965) 103-111. 2 Borum, P. R., Variation in tissue carnitine concentrations with age and sex in the rat, Biochem. J., 176 (1978) 677-681.
3 Carroll, J. E. and Elkin, D. J., A possible role for carnitine in neonatal brain, Ann. Neurol., 6 (1979) 183-184. 4 Chajek, T., Stein, O. and Stein, Y., Pre- and postnatal development of iipoprotein lipase and hepatic triglyceride hydrolase activity in rat tissues., Atherosclerosis, 26 (1977) 549-561.
800
xx, E
400"
g 0 '0
100
Post-natal Age (days)
Fig. 1. Postnatal changes in the concentration of total carnitine, free carnitine, short-chain acylcarnitine and long-chain acylcarnitine in female rat brain. The concentration of carnitine is expressed as nmol/g wet weight of tissue. 0 , total carnitine; &, short-chain acylcarnitine; A, long-chain acylcarnitine; O, free carnitine. The bars shown for total and short-chain acylcarnitine indicate the value of the S.E.M. The S.E.M. values for long-chain acylcarnitine and free carnitine are too small to be included. The values for the S.E.M. varied from 6 to 24% of the mean value. The level of significance of values at different ages compared with the 1-day values were determined using Students' t-test. For total carnitine and short-chain acylcarnitine this is indicated as X (P < 0.05), XX (P < 0.01) and XXX (P < 0.001). For long-chain acylcarnitine and free carnitine the level of significance at 1,5 and 10 days compared with the values at 96 days was P < 0.025. The number of tissue samples, animals and litters used at each age was: 1 day, 5 tissue samples (combining brains of two animals from the same litter for each sample) from 4 litters; 5 days, 9 tissue samples (two brains for each sample) from 4 litters; 10 days, 16 animals from 8 litters; 15 days, 6 animals from 3 litters; 20 days, 19 animals from 6 litters; 30 days, 6 animals from 6 litters; 96 days, 3 animals from 3 litters.
384 5 Foster, P. C. and Bailey, E., Changes in hepatic fatty acid degradation and blood lipid and ketone body content during development of the rat, Enzyme, 21 (1976) 397-407. 6 Fritz, I. B., Carnitine and its role in fatty acid metabolism, Advanc. Lipid Res., 1 (1963) 285-336. 7 Hahn, P. and Skala, J. P., The role of carnitine in brown adipose tissue of suckling rats, Comp. Biochem. Physiol.. 51B (1975) 507-515. 8 Hahn, P., Secombe, D. W. and Towell, M. E., The perinatal role of carnitine. In M. Monset-Couchard and A. Mintowski (Eds.), Physiological and Biochemical basis for Perinatal Medicine, Karger, Basel, 1981, pp. 187-198. 9 Hawkins, R. A., Williamson, D. H. and Krebs, H. A., Ketone-body utilization by adult and suckling rat brain in vivo, Biochem. J., 122 (1971) 13-18. 10 Karpata, G., Carpenter, S., Engel, A., Waiters, G., Allen, J., Rothman, S., Klassen, G. and Mamer, O. A., The syndrome of systemic carnitine deficiency - - clinical morphological and pathophysiological features, Neurology, 25 (1975) 16-24. 11 Marquis, N. R. and Fritz, I. B., The distribution of carnitine, acetylcarnitine and carnitine acetyltransferase in rat tissues, J. biol. Chem., 240 (1965) 2193-2196.
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