Brain Research, 119 (1977) 181-187 © Elsevier/North Holland Biomedical Press, Amsterdam - Printed in The Netherlands

181

M A T U R A T I O N A L C H A N G E S OF A M I N O ACID C O N C E N T R A T I O N IN C E R E B R O S P I N A L F L U I D OF T H E RAT

ROWENA K. KOROBKIN and ROBERT W. P. CUTLER Department of Neurology, Stanford Medical School, Stanford, Calif. 94305 (U.S.A.)

(Accepted April 27th, 1976)

SUMMARY The cisternal spinal fluid (CF), plasma, and brain stem concentrations of 6 amino acids were measured in suckling rats between birth and one month of age. Each of the amino acids studied had its distinctive maturational pattern of CF concentration. Presumably these amino acids have separate mechanisms of transport from CF, which mature at different times. The changes in CF amino acid concentration with maturation were independent of the changes in plasma or brain stem amino acid concentration. Thus the CF/plasma ratios or CF/brain stem ratios in these young animals are age dependent. Age dependence of CSF amino acid concentration or CSF/plasma amino acid ratios must be considered when correlating abnormal CSF/plasma ratios of an amino acid to abnormal cerebral amino acid concentration in young infants.

INTRODUCTION The investigation of human metabolic encephalopathies is hindered by the difficulties of obtaining brain tissue for study during life. Normally, there is a dynamic equilibrium of concentration of metabolites among blood, brain and cerebrospinal fluid (CSF). This equilibrium has led numerous investigators to measure the concentration of a metabolite in CSF and to draw inferences concerning its concentration and turnover in brain. Such an approach is qualitative at best and is complicated by a number of factors. Most important are the regional variation in metabolite concentration in CSF 11, and the presence of transport mechanisms of varying capacity among regions of the CSF compartments s. These points have beeen repeatedly illustrated in studies of catecholamine metabolism in CSFa,0,16. There are many familial amino acidopathies in which transport, excretion, or metabolism of one or several amino acids are abnormal. Clinical neurological ab-

182 normalities are frequently present in infancy. It is reasonable to suppose that the concentration of amino acids in brain may be reflected more precisely by their conce,1tration in CSF than in plasma. For example, patients with glycine encephalopathy have elevated brain and CSF glycine concentrations without corresponding elevation in plasma or urine glycine concentrations while other patients with hyperglycinemia but without cerebral symptoms have normal brain and CSF glycine concentrations ~3. The purpose of this investigation was to define the relationships among blood, brain and CSF amino acids during maturation of the rat. The marked postnatal changes in brain amino acid content in this species provided a natural condition for examining whether similar changes occur in adjacent CSF. MATERIALS AND METHODS Unlabeled dansyl chloride (1-dimethylaminonaphthalene-5-sulphonyl chloride), 10 ~ w/v in acetone, was purchased from the Pierce Chemical Company (Rockford, I11.), and uniformly labeled [SH]dansyl chloride (spec. act., 2.49 Ci/mmole) was purchased from the New England Nuclear Corporation (Boston, Mass.). Sprague-Dawley rats with known time of birth were allowed to suckle freely until 5-10 min before sacrifice when they were anesthetized with 30--50 mg/kg intraperitoneal pentobarbital. Mothers were fed standard rat chow pellets supplied by Feedstuff Products, San Francisco, Calif. Samples of cisternal spinal fluid (CF), 3-5 #1, were obtained by cisternal puncture using the shaft of a 22-gauge needle filed to a blunt tip and attached to a 10 cm length of polyethylene tubing, internal diameter 0.023 in. After cisternal puncture, the animals were decapitated and the heads rapidly frozen in acetone-solid CO2. Blood samples were collected in heparinized capillary tubes from the bleeding carotid arteries. Frozen brain was removed from the skull. A transverse slice of medulla (5-10 mg) was obtained and weighed frozen. This section was chosen to represent brain adjacent to the CF sampled.

Amino acid assay

CF was mixed with equal volumes of 0.05 M sodium bicarbonate buffer, pH 9.0, and of acetone, and then frozen at --20 °C for at least 1 h. Plasma was deproteinized with 5 vol. of acetone at --20 °C for at least 1 h, centrifuged for 30 min at 3000 x g, and the supernate was mixed with an equal volume of bicarbonate buffer. Brain tissue was homogenized by hand in 20 vol. of bicarbonate buffer, centrifuged for 15 min at 7000 × g, and the supernate was mixed with an equal volume of acetone and stored at --20 °C for at least 1 h. Amino acids were then assayed using the dansylation procedure of Briel et al. 2 as modified by Franklin et al. s. Six of the 7 amino acids detected in highest concentration in cisternal fluid were studied. Alanine, although detected consistently, was difficult to separate from dansylamine and was therefore not included in the analysis of data. The Student t-test was used for statistical analysis.

183 RESULTS

Concentration of amino acids in newborn CF (Table 1) The concentration of the 6 amino acids studied in CF of animals less than 2 h of age (newborn) is shown in Table I, and compared with adult values reported by Franklin et al. s. All of the amino acids studied in the CF were higher in the newborn period than in the adult. Taurine, but not the other amino acids, showed a significant variability in newborn concentration from one litter to another, while animals within a litter had similar concentrations. The plasma concentrations of taurine did not show such a variability. This litter to litter variability disappeared by one day of age. Three amino acids which were present in high concentration in brain could not be evaluated in CF. Gamma-aminobutyric acid (GABA), although present in brain stem, was not detected in CF. Aspartic acid and glutamic acid were inconsistently detected in very small quantities throughout life (up to 9 nmoles/ml for aspartic and 23 nmoles/ml for glutamic acid), even though brain stem concentrations of the former increased 4-fold between birth and adulthood. Changes in CF concentration with maturation (Table I) The pattern of maturation of CF concentration varied among the amino acids. Glutamine, lysine and ornithine concentrations fell significantly in the first day of life. Glutamine concentration then returned towards newborn levels and remained relatively constant through the first month of life; lysine concentration continued to fall; and ornithine concentration returned towards newborn levels for one week before falling again. Glycine and taurine concentrations remained elevated initially and then fell to adult levels during the second week of life. Serine concentration increased significantly over the first week of life and then began to fall at two weeks. Relationship between CF amino acid concentration and plasma and brain stem amino acid concentrations (Fig. 1) Ratios were calculated as means of the ratios for individual experimental animals at each age rather than the ratios of the mean vaJues for each age. (a) CF/plasma. The ratio of CF/plasma changed with age and the pattern of this change was different for the different amino acids. Glycine, for example, had a nearly constant CF/plasma ratio from birth to one month of age, while the taurine CF/plasma ratio increased 3-fold in the first days of life, and the serine CF/plasma ratio decreased in the first day of life. (b) CF/brain stem. The ratio of CF/contiguous brain stem changed with age differently for each of the amino acids. Examples of this variability are as follows: the glycine CF/brain stem ratio decreased by two weeks of age; the taurine ratio remained constant until two weeks and then increased; and the glutamine ratio remained nearly constant from birth through the first month of life.

1.84 4.71 1.86 0.45 10.2 0.27

720 1452 473 782 1285 95

188 887 34 339 108 34

± ± 55± 5-

5± 5555-

146 166 71 141 149 18

20 94 3.3 32 12 3.0

0.23 0.37 0.16 0.04 0.98 0.04

i -4555±

3.57 5.23 3.79 0.50 14.2 0.27

1407 1171 511 270 1004 144

262 478 42 114 119 21.5

± 55555-

5: ± ± 555-

0.33* 0.53 0.43* 0.04 0.52 0.03

336* 145 127 24* 149 66

± 81 5- 74* 5- 8.2 J- 4.6* 5- 24 5- 3.7*

1 day (8,4)

1.87 4.06 3.03 0.35 8.93 0.29

1014 1138 307 445 574 173

278 696 48 112 94 31

31" 67** 23 9* 25 3.3

-4- 0.25** 5- 0.63 5- 0.28* 5- 0.05 5- 1.0"* 5- 0.03

5- 141 5- 187 5- 37 -4- 26* 5- 59* ± 25

-+± ± 5± 5-

4 days (7,2)

2.43 5.27 3.69 0.37 11.6 0.34

1491 1373 363 275 851 174

297 741 42 76 138 34

± 55555-

55555±

193" 267 49 36* 153 22

37* 93 15 9.7* 32 3.7**

0.49 1.0 0.68* 0.07 1.3 0.08

-t± 55± 5-

7 days (6,2)

* P -< 0.05, concentrations significantly different from those of the newborn. ** P -< 0.05, concentrations significantly different from those at one day. *** Values taken from Franklin et al. 8.

Serine Glutamine Glycine Lysine Taurine Ornithine

Brain stem (#mole/g 5- S.E.)

Serine Glutamine Glycine Lysine Taurine Ornithine

Plasma (nmole/ml ± S.E.)

Serine Glutamine Glycine Lysine Taurine Ornithine

Cisternalfluid (nmole/ml 5- S.E.)

Newborn (10,2)

Numbers in parentheses indicate the n u m b e r of animals, the n u m b e r of litters.

Values of amino acid concentration at various ages

TABLE I

2.63 4.86 3.86 0.41 5.87 0.34

1082 1203 410 369 388 129

225 599 22 66 66 15

± 5555±

555± 5±

0.28 0.81 0.41" 0.06 0.42** 0.05

178 273 117 81" 50* 31

± 42 ± 107 ± 3.9* 5_ 14" 5- 4.8* 5- 2.0*

14 days (7,2)

1.94 3.84 3.16 0.40 1.86 0.14

329 779 179 157 349 22

96 645 20 57 57 4.6

5_ 0.32 ± 0.54 ± 0.33* ± 0.05 5- 0.10" 5- 0.02*

± 75* ± 313 5- 71 5- 89* ± 64* -5- 10"

± 21" ± 128 5- 4.5* 5- 13" 5- 7.1" 51.6"

29-33 days (4,2)

0.75 3.39 4.02 0.41 2.00

196 619 221 290 432

79 863 20 120 66

55± ± ±

5± ± ± ±

± ± ± 55-

0.13 0.33 0.18 0.03 0.2

14 42 13 32 38

12 98 1 13 6

Adult*** (8)

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AGE IN DAYS

Fig. 1. Maturation from birth to one month of age of the relationship between CF/plasma (open squares), CF/brain stem (filled squares), and brain stem/plasma (open circles) for each of the 6 amino acids studied. The newborn ratio is given as 100 ~ and subsequent ratios (means of the ratios of 4-9 animals at each age) are expressed as per cent of the newborn value. The asterisks indicate a significant (P < 0.05) difference from the newborn value. DISCUSSION The m a t u r a t i o n a l change o f C S F a m i n o a c i d c o n c e n t r a t i o n has n o t been well studied. T h e r e are n o a n i m a l studies r e p o r t e d a n d the h u m a n values r e p o r t e d are frequently those o f patients with n e u r o l o g i c a l disease. T h e r e is a suggestion o f a fall in serine c o n c e n t r a t i o n between infancy a n d a d u l t h o o d in the d a t a o f D i c k i n s o n a n d

186 Hamilton 5, and Perry et al. 14 noted that glutamine concentrations in the CSF of infants are 'somewhat lower' than in adults. Homocarnosine concentration has been shown to fall between birth and adulthood ~2. We found distinct maturational patterns of CSF concentration for each of the amino acids studied. The 6 amino acids studied belong to 4 transport classes: dibasic, neutral, imino and fl-amino acids. Ornithine and lysine, both dibasic amino acids, showed a significant fall in CF concentration between birth and one day of age. Although lysine concentration then remained low, ornithine increased again to newborn values by the end of the first week and then decreased. The neutral amino acids, glutamine, serine and glycine, showed no consistent pattern of maturational change. There were not enough amino acids of any class to show relationships between maturational patterns of CSF amino acid concentration and transport categories; the data we do have suggest that this is not the case. The relationship between the CSF concentration of an amino acid and its plasma or brain concentration is of considerable clinical interest. One would like to infer abnormalities in brain concentration from abnormalities in CSF concentration or CSF/plasma ratios. In studies attempting to correlate brain tryptophan metabolism with CSF concentrations of the amine and its metabolites, Modigh showed that rats fed a high tryptophan diet had elevated concentrations of tryptophan and 5-HIAA in whole brain as well as CSF, with significant correlations between brain and CSF concentrations 1°. On the other hand, Bulat and ~ivkovit~ showed that the 5-HIAA concentration in lumbar CSF is derived from spinal cord rather than cisternal CSF or blood 3. Even if there were a correlation between CSF and brain in the adult animal, one must ask whether the relationship changes with development. In our study there did not appear to be any consistent relationship among the 3 compartments, blood, brain and CSF during normal maturation in the rat. For example, in the first day of life, brain stem and plasma ornithine remained constant or increased slightly, while in spinal fluid adjacent to the brain stem, ornithine concentration fell significantly. It appears that the maturation of regulatory mechanisms of amino acid concentration in the CSF is independent of that in plasma and brain. Similarly, Ferguson and Woodbury found the timing of developmental changes in Na, K and CI concentrations in rat CSF to be unrelated to plasma changes 6. This lack of correlation between CSF and plasma maturational changes has been shown for glucose and protein concentration in the chick embryo 15, for K, Mg and Ca in the fetal monkey ~, and for Na, CI and urea in the fetal pig 7. This indicates the importance of having age matched controls when trying to determine an abnormality of CSF/ plasma ratio in a disease state. For any given age, there may be a normal ratio between any two of the 3 compartments, but disturbance in one compartment would not necessarily be reflected in another. If there is active transport of the amino acid out of the CSF, its CSF/ plasma ratio might not be abnormal in the steady state unless the transport mechanisms for effiux from CSF were saturated, even if the brain were producing abnormal quantities. The CSF/plasma ratio would be more likely to be elevated with abnormal brain metabolism if there is no active transport of the amino

187 acid o u t o f the C S F . This is likely for g l u t a m i n e where the C S F / p l a s m a r a t i o a p p r o a c h es unity at all ages studied. It is o f interest t h a t patients with h e p a t i c e n c e p h a l o p a t h y have elevated C S F / p l a s m a g l u t a m i n e ratios 4. A l t h o u g h the finding o f an a b n o r m a l C S F / p l a s m a ratio o f an a m i n o acid m a y be helpful in evaluating clinical disease (e.g., glycine e n c e p h a l o p a t h y l a ) , a n o r m a l r a t i o m a y n o t i m p l y n o r m a l b r a i n m e t a b o l ism. ACKNOWLEDGEMENT This w o r k was s u p p o r t e d b y U.S. Public H e a l t h Service G r a n t s 7 F32 HD05058 a n d NS-12079.

REFERENCES 1 Bito, L. Z. and Myers, R. E., The ontogenesis of haematoencephalic cation transport processes in the rhesus monkey, J. Physiol. (Lond.), 208 (1970) 153-170. 2 Briel, G., Neuhoff, V. and Maier, M., Microanalysis of amino acids and their determination in biological material using dansylchloride, Hoppe-Seyler's Z. physiol. Chem., 353 (1972) 540-553. 3 Bulat, M. and ~ivkovi6, B., Origin of 5-hydroxyindoleacetic acid in the spinal fluid, Science, 173 (1971) 738-740. 4 Caesar, J., Levels of glutamine and ammonia and the pH of cerebrospinal fluid and plasma in patients with liver disease, Clin. Sci., 22 (1962) 33-41. 5 Dickinson, J. C. and Hamilton, P. B., The free amino acids of human spinal fluid determined by ion exchange chromatography, J. Neurochem., 13 (1966) 1179-1187. 6 Ferguson, R. K. and Woodbury, D. M., Penetration of [14C]inulin and [14C]sucrose into brain, cerebrospinal fluid and skeletal muscle of developing rats, Exp. Brain Res., 7 (1969) 181-194. 7 Flexner, L. B., Changes in the chemistry and nature of cerebrospinal fluid during fetal life in the pig, Amer. J. PhysioL, 124 (1938) 131-135. 8 Franklin, G. M., Dudzinski, D. S. and Cutler, R. W. P., Amino acid transport into the cerebrospinal fluid of the rat, J. Neurochem., 24 (1975) 367-372. 9 Garelis, E., Young, S. N., Lal, S. and Sourkes, T. L., Monoamine metabolites in lumbar CSFthe question of their origin in relation to clinical studies, Brain Research, 79 (1974) 1-8. 10 Modigh, K., The relationship between the concentrations of tryptophan and 5-hydroxyindoleacetic acid in rat brain and cerebrospinal fluid, J. Neurochem., 25 (1975) 351-352. 11 Moir, A. T. B., Ashcroft, G. W., Crawford, T. B. B., Eccleston, D. and Guldberg, H. C., Cerebral metabolites in cerebrospinal fluid as a biochemical approach to the brain, Brain, 93 (1970) 357-368. 12 Perry, T. L., Hansen, S., Stedman, D. and Love, D., Homocarnosine in human cerebrospinal fluid: an age-dependent phenomenon, J. Neurochem., 15 (1968) 1203-1206. 13 Perry, T. L., Urquhart, N., MacLean, J., Evans, M. E., Hansen, S., Davidson, A. G., Applegarth, D. A., MacLeod, P. J. and Lock, J. E., Glycine accumulation in brain in nonketotic hyperglycinemia, New Engl. J. Med., 292 (1975) 1269-1273. 14 Perry, T. L., Hansen, S. and Kennedy, J., CSF amino acids and plasma-CSF amino acid ratios in adults, J. Neurochem., 24 (1976) 587-589. 15 S e d l ~ k , J., Some basic chemical components of the cerebrospinal fluid in developing chick embryos, Physiol. bohemoslov., 24 (1975) 305-310. 16 Vogt, M., Metabolites of cerebral transmitters entering the cerebrospinal fluid; their value as indicators of brain function. In H. F. Cserr, J. D. Fenstermacher and V. Fencl (Eds.), Fluid Environment of the Brain, Academic Press, New York, 1975, pp. 225-236.