Clinica Chimica Acta, 92 (1979) 65-72 @ Elsevier/North-Holland Biomedical Press

CCA 9901



*, E.H.F.





and G.M. ABER

Departments of Neurology and Nephrology, Medical Research Unit, North Staffordshire Hospital Centre, Hartshill, Stoke-on-Trent, ST4 7NY (U.K.) (Received




Summary The concentration of free amino acids in the plasma and lumbar CSF of 11 patients with steady-state chronic renal failure has been measured and the CSF : plasma concentration ratios calculated. The results have been compared with the corresponding data from 37 control subjects. In renal failure, elevation of the mean plasma concentration of total amino acids and a reduction in the ratio of essential to total amino acids have been found. Whereas some individual plasma amino acid concentrations in renal failure were higher than normal, others were lower. Striking abnormalities of the CSF amino acid concentration have been observed. Some amino acids have shown similar patterns of abnormality in both CSF and plasma, whereas in the case of others, the changes have been restricted to either CSF or plasma. Significant variations from normal of the CSF : plasma concentration ratios were observed for four amino acids.

Introduction Previous studies have shown major quantitative and minor qualitative differences between the free amino acid content of plasma and cerebrospinal fluid (CSF) in normal subjects [1,2]. It has also been established that the plasma amino acid profile is abnormal in patients with chronic renal insufficiency, with low concentrations of the essential amino acids, high concentrations of non essential amino acids and a high phenylalanine/tyrosine ratio [3,4]. Whether * Present address: Department of Neurology, Leiceeter Royal Infirmary. Leicester. LEl 6WW. U.K. ** Correspondence should be addressed to: Dr. E.H.F. McGale. North Stafford-e Medical Institute, Medical Research Unit, Her&shill, Stoke-on-Rent. ST4 7NY. U.K.


these changes in plasma amino acid concentrations produce any alteration in the CSF amino acid profile is, however, not known. The present investigation has been designed to study the CSF amino acid profile of patients in steady-state renal failure and to relate the changes in total and individual CSF amino acid concentrations to those occurring in plasma. Patients and methods Eleven patients (6 males, ‘5 females; mean age 045.4 year) with steady-state chronic renal failure were studied. The mean blood urea and serum creatinine concentrations were 40.5 mmol/l and 1070 pmol/l respectively. The criteria of steady-state were that three successive measurements of blood urea and serum creatinine were stable over a period of not less than seven days while on a constant daily protein intake of 40 to 50 g. None of the patients in the study were being treated by haemodi~ysis. Supplemen~tion of the diet with fat and carbohydrate was encouraged to maintain a high total calorie intake of 25003000 calories/day. Seven patients were receiving therapy for hypertension which is known not to influence plasma amino acid concentrations (McGale and Aber, 1973, unpublished observations). Ten of the patients had clinical or elec~ophysiolo~c~ evidence of a peripheral neuropathy. CSF exudation formed part of the investigation of the neuropathy in sev,en of these patients and in the remaining four, lumbar puncture was performed with the informed consent of the individual. All eleven patients had normal CSF cytology, glucose, total protein concentrations and serology. Thirty seven subjects (17 males, 20 females; mean age 35.6 years) who had been demonstrated to have no neurolo~c~ or metabolic abno~alities formed a control group and have been reported in detail elsewhere [ 21. Collection of specimens

After a 12 h overnight fast, CSF was obtained from each subject by routine lumbar puncture technique and 10 ml of blood were collected simultaneously from the ~~cubit~ vein into bottles confining lithium heparinate. Treatment of specimens and amino acid analysis

Immediately after collection, the specimens of CSF and plasma were deproteinised with 3% sulphosalicyclic acid. Details of this procedure and of the subsequent andysis using a six sample dual column amino acid analyser and integrator (model JLC 6AH, Jeol Ltd., 1418, Nakagami, Tokyo) have been reported previously [ 21. Glutamine and glutamic acid were measured in a separate ahquot of each specimen by .a double enzymatic NAD dependent system [5]. The coefficient of variation.of this method was 2.1% for CSF and 1.9% for plasma. Stutisticai anul~sis of results

The individual and total amino acid concen~ations of plasma and CSF and the CSF : .plasma ratios of individual amino acids were compared with the control values using the Wilcoxon rank sum test. Inter-relationships of individual amino acid concentrations in CSF and plasma were assessed by measurement of the linear (product-moment) correlation coefficient.


Results Tables I and II show the plasma and CSF concentrations respectively of those amino acids which differed significantly from normal in the patients with steady-state chronic renal failure. For all other amino acids measured, no significant variations from the control values were observed in chronic renal failure. Plasma amino acid concentrations In the patients with renal failure, the mean plasma concentration of total amino acids was 4060 2 909 pmol/l compared with 3564 k 891 pmol/l in the control subjects (0.05 > p > 0.02). Although the mean concentration of essential amino acids did not differ from normal, the ratio of essential to total amino acids was lower in the renal (19.3 k 3.3%) than in the control group (27.3 + 3.l%;p < 0.01). The plasma concentrations of phosphoserine, phosphoethanolamine, proline, glycine, cystine, l-methylhistidine and 3-methylhistidine were elevated significantly in the renal patients @ < 0.005-0.05) and those of valine and tyrosine were reduced Cp < 0.01). The mean phenylalanine : tyrosine ratio was 1.34 t 0.22 in the renal patients compared with 0.88 k 0.14 in the control subjects (p < 0.01) and the mean valine : glycine ratios in the two groups were 0.53 ? 0.21 and 1.15 k 0.45 @ < 0.01) respectively. CSF amino acid concentrations Although the mean CSF concentrations of total, non-essential and essential amino acids did not differ significantly in the renal and control subjects, the CSF concentrations of certain amino acids (phosphoserine, phosphoethanol-


Amino acid concentration (pmoI/l) Control subjects (ref. 2) (n = 37) Mean

Phosphoserine Phosphoethanolamine Proline Glycine VaIine Cystine Tyrosine 1-Methylhistidine 3-Methylhistidine * Insufficient for quantitation.

8.3 5.1 281.6 282.7 308.6 123.7 73.0 * *

Steady-state renal failure patients (n = 11)

RenaIs compared with controls, P value


5.7 3.3 191.3 101.6 116.0 50.0 26.3 *




41.1 14.4 479.2 451.0 209.7 314.1 44.9 30.2 61.9

17.1 6.5 343.4 184.9 61.4 133.6 10.2 14.7 38.1

p > 0.02 < 0.01 < 0.01 < 0.01 i 0.01 < 0.005 0.01

=O.Ol 0.05 > p > 0.01 O.l >0.2 >0.2 >0.05 >0.5 0.05 >O.l 0.01 >0.06 0.02

> p > 0.001 > P > 0.025 > p > 0.026 > P > 0.005

> p > 0.026 > p > 0.005 > p > 0.01


Phosphoserine Glutamine P-Aminobutyric Omithine

Concentration ratios


Control subjects (n = 37)

Renal patients (n = 11)





Renal compared with control, P value

0.58 0.86 0.14 0.06

0.34 0.12 0.07 0.02

0.22 0.72 0.22 0.08

0.11 0.12 0.17 0.03


of phosphoserine and glutamine lower (p < 0.01). Insufficient figures were available to permit any comparison of the ratios of l-methylhistidine and 3-methylhistidine owing to the fact that these are either absent or present in only trace amounts in normal CSF. No significant, differences were found between male and female renal patients for any amino .acid CSF : plasma ratio. Dicussion The present study of patients with steady-state chronic renal failure on a controlled dietary protein intake has confirmed previously published observations concerning the disturbance of plasma amino acids associated with renal failure [ 3,4]. In addition, marked abnormalities of the CSF amino acid profile have been demonstrated which as far as we are aware have not been reported previously. Whereas some of the changes observed in’CSF amino acids appear to parallel those in the pIasma, the overall CSF amino acid pattern in patients with chronic renal failure is by no means a simple reflection of changes of plasma amino acid concentration. Indeed, three patterns of abnormality were noted. Firstly, where a significant increase or decrease in the concentration occurred in both plasma and CSF; secondly, where significant changes in the concentration of certain amino acids were restricted to the CSF and thirdly, where significant changes occurred in plasma alone. Although the physiological determinants of these findings are speculative, one must include a consideration of differing concentration gradients between plasma and CSF, active transport mechanisms at the blood-brain or blood-CSF interfaces and the metabolism of amino acids by the central nervous system itself. Where a positive correlation between the plasma and CSF concentration of an individual amino acid was noted, one could imply that the plasma was a major determinant of its CSF concentration. Such a correlation was found for 15 amino acids in the control subjects but for only 6 in the patients with chronic renal failure. Although a further 7 amino acids in patients with renal failure had abnormal concentrations in both plasma and CSF only 1 (l-methylhistidine) had a positive correlation between its plasma and CSF concentration, whereas, in the control subjects, 3 of these 7 amino acid8 had shown a positive


correlation between. their plasma and CSF concentration. From these observations it is clear, therefore, that the plasma amino acid concentration itself is by no means the only factor determining the CSF amino acid profile. Animal experiments suggest that the transport of amino acids at the interfaces between blood and brain and blood and C$F plays a major role in maintaining the separate identities of the free amino ‘acid pools in these various biological compartments. In each pool, amino acids are in dynamic equilibrium with many that are undergoing rapid metabolism and although exchange between brain and CSF can occur fairly freely, it is restricted at the blood-brain and blood-CSF barriers by b&directional. transport systems which are carrier mediated, stereospecific and have ‘saturable -and non-saturable bomponents [6,7]. Separate systems hav_e been shown to exist for neutral, basic and acidic amino acids [8-101. Since the plasma and the CSF abnormalities observed in the present study involve amino acids which have both differing transport systems and different rates of entry to the brain [ll], no systematic disturbance of any particular transport mechanism can account for all the changes noted. Nevertheless, it would seem highly likely that interference with the normal amino acid transport mechanism is likely to occur in the “uraemic” state. This concept receives considerable support from previous studies designed to examine disturbance of bromide, inulin and sucrose handling at blood-brain and blood-CSF interfaces [ 12,131. The aetiology of the neurological complications of chronic renal failure is as yet unexplained [14] and it is not possible from our present findings to say whether they are related to the CSF and/or plasma abnormalities. It would not seem unreasonable, however, to suggest that clinical neurological syndromes associated with the chronic renal failure might be attributable to abnormalities of amino acid metabolism in neural tissue, especially if the particular amino acids concerned are putative neurotransmitters or their precursors e.g. glycine and tyrosine respectively. Brain catecholamine and serotonin synthesis may be regulated by the cerebral concentration of their precursor amino acids [E] which in turn are governed by their plasma levels [ 161, and recently, abnormalities of CSF metabolites of serotonin and dopamine have been observed in human uraemic encephalopathy [ 171. This hypothesis complements the suggestion that defective “sink action” of CSF for acid metabolites may be implicated in the pathogenesis of the encephalopathy seen in the acutely uraemic rat

CW. Acknowledgements We are grateful to the Medical Research Council and the Research Committee of the West Midlands Regional Health Authority for financial support. References 1 Perry. T.L.. Hansen, S. and Kennedy, J. (1975) J. Neurochem. 24.587-589 2 McGaIe. E.H.F., Pye. I.F.. Stonier, C., Hutchinson. E.C. and Aber, G.M. (1977) 291-297 3 McGaIe. E.H.F., Pickford. J.C. and Aber, G.M. (1972) CIin. Chbn. Acta 38.395-403 4 Kopple, J.D. and Swendseid. M.E. (1975) Kidney Int. 7.64-72

J. Neurochem.


72 5 Pye, I.F.. Stonier, C. and MaGale. E.H.F. (1978) Anal. Chem. 50.961-953 6 Laitha, A. (1974) Aromatic amino acids in the brain (CIBA Foundation SYmPosium. No. 22). PP. 25-41, Ebevier, London 7 Lorenso, A.V. (1974) Fed. Proc. 33.2079-2086 8 Rapoport, S.I. (1976) Blood-brain Barrier in Physiology and Medicine, Raven Press. New York 9 Pardridge. W.M. (1977) J. Neurochem. 28.103-108 10 Oldendorf, W.H. and Ssabo. J. (1976) Am. J. Physiol. 230.94-98 11 Barios, G., Daniel, P.M., Moorhouse, S.R. and Pratt, O.E. (1976) J. Physiol. (London) 246.539-548 12 Freeman, R.B., Sheff. M.F., Maher, J.F. and Schreiner. G.E. (1962) Ann. Intern. Med. 66.233-240 13 Fisbman. R.A. and Rashin. N.H. (1967) Arch. Neurol. 17.10-21 14 Tyler, H.R. (1975) Kidney Int. 2.188-193 15 Femstrom, J.D. and Wurtman. R.J. (1972) Science 178.414-416 16 Daniel, P.M., Moorhouse. S.R. and Pratt, O.E. (1976) Psychol. Med. 6,277-286 17 Sullivan, P.A., Mumaghan, D., CaBagban. N.. Kantamaneni, B.D. and Curson, G. (1978) J. NeuroI. Neurosurg. Psych&r. 41.581-688 18 Mann, J.D. and Bass, N.H. (1975) Trans. Am. Neurol. Assoc. 100,218-220

Studies of cerebrospinal fluid and plasma amino acids in patients with steady-state chronic renal failure.

65 Clinica Chimica Acta, 92 (1979) 65-72 @ Elsevier/North-Holland Biomedical Press CCA 9901 STUDIES OF CEREBROSPINAL FLUID AND PLASMA AMINO ACIDS I...
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