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Positive correlation between contamination by blood and amino acid levels in cerebrospinal fluid of the rat
212
Neurvscience Letters. 69 (1986) 212 2 t > Elsevier Scientific Publishers Ireland I,I¢I
NSL 04113
POSITIVE CORRELATION BETWEEN C O N T A M I N A T I O N BY B L O O D AND AMINO ACID LEVELS IN CEREBROSPINAL FLUID OF THE RAT
M A L T E E. K O R N H U B E R * , J O H A N N E S K O R N H U B E R , A N S E L M W. K O R N H U B E R and G E R T M. H A R T M A N N
Department q/Neurology, Universi O' of UIm, Steinhgvelstr. 9, D-7900 Ulm ( F.R.G.) (Received April 16th, 1986: Revised version received May 22nd, 1986; Accepted May 23rd, 1986)
Key words: cerebrospinal fluid - contamination by blood - amino acid - neurotransmitter - H P L C rat The degree of contamination by blood in macroscopically clear cerebrospinal fluid (CSF) from the rat was assessed by the red blood cell count. A m i n o acid concentrations in the same samples were determined using high performance liquid chromatography. A significant positive correlation between the number of erythrocytes and amino acid concentration was found for alanine, asparagine, aspartate, citrulline, glutamate, glycine, phenylalanine and taurine but not for glutamine, histidine, isoleucine, leucine, lysine, methionine, ornithine, serine, threonine, tyrosine and valine. The difference in amino acid concentration between samples that were or were not contaminated by blood was as m u c h as one order of magnitude for aspartate, glutamate, glycine and taurine; the concentrations o f these amino acids were highly correlated with the erythrocyte counts (r = 0.87, P = 0.000002 for glutamate). The results suggest that macroscopical inspection is often not sufficient to judge contamination by blood in the CSF.
Cerebrospinal fluid (CSF) samples are frequently contaminated by blood. Since many substances common to both media are present in much higher concentrations in blood as compared to CSF, the degree of contamination determines the validity of measured CSF solute concentrations. Usually the degree of contamination is judged subjectively by macroscopical inspection. However, the perceptual threshold for visual detection of contamination by blood varies widely [2] and corresponds to a minimum erythrocyte count of about 2000-3000/lal [2, 6]. Our results demonstrate that even visibly undetectable levels of contamination by blood significantly increase the concentration of several amino acids in rat CSF. Amino acid concentrations were measured in CSF samples taken from 24 male 250 g CHBB:THOM rats (Thomae, Biberach, F.R.G.) housed in groups of two per cage with free access to food and water. To collect samples, anesthetized animals (chloralhydrate 3.5%, 2.5 ml, i.p.) were mounted in a stereotaxic frame and the atlantooccipital membrane was carefully exposed to allow access to the cisterna magna. A total of about 100 Ial CSF were removed from each animal using a microsyringe fixed to a micromanipulator (for a more detailed description of the CSF collection technique see ref. 1). in a 10-1al traction of each CSF sample the degree of contamination by *Author for correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
213
blood was measured as the number of erythrocytes detectable with a Fuchs-Rosenthai chamber. The remaining 90 gl of each sample were immediately deproteinized by microcollodium filtration (Sartorius, G6ttingen, F.R.G.) and stored at - 8 0 'C until analysis. The maximum interval between CSF sampling and freezing was 5 min. The concentrations of 19 amino acids were determined by high performance liquid chromatography (HPLC) as previously described [7]. In 9 CSF samples no erythrocytes were detected. In the remaining 15 samples the contamination varied between 5 and 2400 erythrocytes/I, tl. In Table I amino acid concentrations are given for those CSF samples in which no erythrocytes were detected. There was a significant positive correlation between the erythrocyte count and the CSF concentrations of alanine, asparagine, aspartate, citrulline, glutamate, glycine, phenylalanine and taurine but not of glutamine, histidine, isoleucine, leucine, lysine, methionine, ornithine, serine, threonine, tyrosine and valine (Table I). The amino acid whose concentration was most strongly correlated with the erythrocyte count was glutamate (r =0.873, P=0.000002). This result is shown in Fig. 1A together with the results for glutamine (Fig. 1B). Since many neurotransmitters are amino acids or amino acid derivatives, their determination in CSF may be useful for the investigation of central neurotransmission. TABLE I A M I N O ACID C O N C E N T R A T I O N IN RAT ('SF Only samples wilh no erythrocytes were used l\)r calculation: values are expressed as nmol/ml. ()n Ihe righl hand sidc the correlation coefficient (r) is given for the relationship between the number of erythrocytes and amino acid concentration. *P < 0.05: **P < 0.01 : ***P < 0.00 I.
Alanme Asparaginc Aspartale Citrulline (}lutanaate Glutamme
Glycinc llistidine lsolcucine [.etlcine Lysme Methionine
Ornilhine Phenylalanine Serine Ta,arine
Threonine Tyrosine Valine
Mean + S.E.M.
r
(n - 9 t
(n
43.89_+ 2.39 3.65 +_ 0.37 0.26 _+ 0.05 2.92_+ (I.I 6 2.56_+ 0.17 317.58 + 18.93 3.62 _+ 0.33 6.80_+ 1.11 1.75-+ 0.13 3.77_+ 0.37 39.17_+ 2.13 2.62 _+ 0.29 2.51+_ 0.13 2.44_+ 0.29 54.21 q- 4.68 14.58+ 1.47 37.33_+ 2.94 8.61 + 0.65 4.27 _+ 0.43
24) 0.507* 0.589** 0.803*** (I.650"** 0.873*** 0.103 0.790*** 0.061 0.347 0.308 0.199 0.289 0.197 0.559** 0.137 0.807*** 0.189 0.080 (I.075
214 Glutamate
r=0873
1)<0 001
Glutamine
r = 0 103 n ~
12
10
!.. ~
.
0
30o1? A
~00
B
100] 1000
20(30
--~
0
1000
2 0 0 0 e r y t h r o c y t e s / ;Jl CSF
Fig. 1. The two scattergrams illustrate the relationship between the erythrocyte count in the CSF and glutamate (A) and glutamine (B) levels, respectively. Values are expressed in nmol/M1, n.s., not significant.
The effect of introducing blood constituents during sample collection or otherwise, must be addressed in studies devoted to this field. Blood contamination of the CSF has usually been assessed by macroscopical inspection, with slightly reddish specimens considered unusable. Our results clearly show that this simple criterion is not adequate for screening unduly contaminated samples. The difference in amino acid concentration in contaminated and uncontaminated samples was as much as one order of magnitude for aspartate, glutamate, glycine and taurine even though all samples appeared macroscopically uncontaminated. Variations in the concentrations of different amino acids may be partly due to reported normal variations in the plasmaCSF gradient (e.g. ref. 4). However, the correlation between the amino acid concentration and the number of erythrocytes in the CSF is higher than could be expected simply on the basis of plasma-CSF gradients. Further factors which might explain our findings could be the high concentration of certain amino acids in blood cells [3, 5] and blood enzymes causing amino acid changes in the CSF. From our results we conclude that the contribution of non-visible contamination by blood to measures of CSF constituents should be assessed at least for substances known to have high blood-CSF gradients. We wish to thank H. Zettlmeissl for the facility to carry out the HPLC measurements. 1 Kornhuber, J., Kim, J.S. and Kornhuber, M.E., CSF sampling techniques. In J.H. Hingtgen, D.H., Hellhammer and G. Huppmann (Eds.), Advanced Methods in Psychobiology, Hogrefe, Toronto, in press. 2 Patten, B.M., How.much blood makes the cerebrospinal fluid bloody?, JAMA, 206 (1968) 378. 3 Perry, T.L. and Hansen, S., Technical pitfalls leading to errors in the quantitation of plasma amino acids, Clin. Chim. Acta, 25 (1969) 53-58. 4 Perry, T.L., Hansen, S. and Kennedy, J., CSF amino acids and plasma-CSF amino acid ratios in adults, J. Neurochem., 24 (1975) 587-589. 5 Soupart, P., Free amino acids of blood and urine in the human. In J.T. Holden (Ed.), Amino Acid Pools, Elsevier, Amsterdam, 1962, pp. 220-262.
215 6 Ylitalo, P., Heikkinen, E.R. and Myllylfi V.V., Evaluation of successive collection of cisternal cerebrospinal fluid in rals, rabbits, and cats, Exp. Neurol., 50 (1976) 330-336. 7 Zettlmeissl, H., Blome, J. and Kornhuber, H.H., A sensitive, fast, durable, highly sensitive method for the determination of amino acids and biogcnic amines in the cerebrospinal fluid and other body fluids and tissues, Arch. hal. Biol., in press.
Neurvscience Letters. 69 (1986) 212 2 t > Elsevier Scientific Publishers Ireland I,I¢I
NSL 04113
POSITIVE CORRELATION BETWEEN C O N T A M I N A T I O N BY B L O O D AND AMINO ACID LEVELS IN CEREBROSPINAL FLUID OF THE RAT
M A L T E E. K O R N H U B E R * , J O H A N N E S K O R N H U B E R , A N S E L M W. K O R N H U B E R and G E R T M. H A R T M A N N
Department q/Neurology, Universi O' of UIm, Steinhgvelstr. 9, D-7900 Ulm ( F.R.G.) (Received April 16th, 1986: Revised version received May 22nd, 1986; Accepted May 23rd, 1986)
Key words: cerebrospinal fluid - contamination by blood - amino acid - neurotransmitter - H P L C rat The degree of contamination by blood in macroscopically clear cerebrospinal fluid (CSF) from the rat was assessed by the red blood cell count. A m i n o acid concentrations in the same samples were determined using high performance liquid chromatography. A significant positive correlation between the number of erythrocytes and amino acid concentration was found for alanine, asparagine, aspartate, citrulline, glutamate, glycine, phenylalanine and taurine but not for glutamine, histidine, isoleucine, leucine, lysine, methionine, ornithine, serine, threonine, tyrosine and valine. The difference in amino acid concentration between samples that were or were not contaminated by blood was as m u c h as one order of magnitude for aspartate, glutamate, glycine and taurine; the concentrations o f these amino acids were highly correlated with the erythrocyte counts (r = 0.87, P = 0.000002 for glutamate). The results suggest that macroscopical inspection is often not sufficient to judge contamination by blood in the CSF.
Cerebrospinal fluid (CSF) samples are frequently contaminated by blood. Since many substances common to both media are present in much higher concentrations in blood as compared to CSF, the degree of contamination determines the validity of measured CSF solute concentrations. Usually the degree of contamination is judged subjectively by macroscopical inspection. However, the perceptual threshold for visual detection of contamination by blood varies widely [2] and corresponds to a minimum erythrocyte count of about 2000-3000/lal [2, 6]. Our results demonstrate that even visibly undetectable levels of contamination by blood significantly increase the concentration of several amino acids in rat CSF. Amino acid concentrations were measured in CSF samples taken from 24 male 250 g CHBB:THOM rats (Thomae, Biberach, F.R.G.) housed in groups of two per cage with free access to food and water. To collect samples, anesthetized animals (chloralhydrate 3.5%, 2.5 ml, i.p.) were mounted in a stereotaxic frame and the atlantooccipital membrane was carefully exposed to allow access to the cisterna magna. A total of about 100 Ial CSF were removed from each animal using a microsyringe fixed to a micromanipulator (for a more detailed description of the CSF collection technique see ref. 1). in a 10-1al traction of each CSF sample the degree of contamination by *Author for correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
213
blood was measured as the number of erythrocytes detectable with a Fuchs-Rosenthai chamber. The remaining 90 gl of each sample were immediately deproteinized by microcollodium filtration (Sartorius, G6ttingen, F.R.G.) and stored at - 8 0 'C until analysis. The maximum interval between CSF sampling and freezing was 5 min. The concentrations of 19 amino acids were determined by high performance liquid chromatography (HPLC) as previously described [7]. In 9 CSF samples no erythrocytes were detected. In the remaining 15 samples the contamination varied between 5 and 2400 erythrocytes/I, tl. In Table I amino acid concentrations are given for those CSF samples in which no erythrocytes were detected. There was a significant positive correlation between the erythrocyte count and the CSF concentrations of alanine, asparagine, aspartate, citrulline, glutamate, glycine, phenylalanine and taurine but not of glutamine, histidine, isoleucine, leucine, lysine, methionine, ornithine, serine, threonine, tyrosine and valine (Table I). The amino acid whose concentration was most strongly correlated with the erythrocyte count was glutamate (r =0.873, P=0.000002). This result is shown in Fig. 1A together with the results for glutamine (Fig. 1B). Since many neurotransmitters are amino acids or amino acid derivatives, their determination in CSF may be useful for the investigation of central neurotransmission. TABLE I A M I N O ACID C O N C E N T R A T I O N IN RAT ('SF Only samples wilh no erythrocytes were used l\)r calculation: values are expressed as nmol/ml. ()n Ihe righl hand sidc the correlation coefficient (r) is given for the relationship between the number of erythrocytes and amino acid concentration. *P < 0.05: **P < 0.01 : ***P < 0.00 I.
Alanme Asparaginc Aspartale Citrulline (}lutanaate Glutamme
Glycinc llistidine lsolcucine [.etlcine Lysme Methionine
Ornilhine Phenylalanine Serine Ta,arine
Threonine Tyrosine Valine
Mean + S.E.M.
r
(n - 9 t
(n
43.89_+ 2.39 3.65 +_ 0.37 0.26 _+ 0.05 2.92_+ (I.I 6 2.56_+ 0.17 317.58 + 18.93 3.62 _+ 0.33 6.80_+ 1.11 1.75-+ 0.13 3.77_+ 0.37 39.17_+ 2.13 2.62 _+ 0.29 2.51+_ 0.13 2.44_+ 0.29 54.21 q- 4.68 14.58+ 1.47 37.33_+ 2.94 8.61 + 0.65 4.27 _+ 0.43
24) 0.507* 0.589** 0.803*** (I.650"** 0.873*** 0.103 0.790*** 0.061 0.347 0.308 0.199 0.289 0.197 0.559** 0.137 0.807*** 0.189 0.080 (I.075
214 Glutamate
r=0873
1)<0 001
Glutamine
r = 0 103 n ~
12
10
!.. ~
.
0
30o1? A
~00
B
100] 1000
20(30
--~
0
1000
2 0 0 0 e r y t h r o c y t e s / ;Jl CSF
Fig. 1. The two scattergrams illustrate the relationship between the erythrocyte count in the CSF and glutamate (A) and glutamine (B) levels, respectively. Values are expressed in nmol/M1, n.s., not significant.
The effect of introducing blood constituents during sample collection or otherwise, must be addressed in studies devoted to this field. Blood contamination of the CSF has usually been assessed by macroscopical inspection, with slightly reddish specimens considered unusable. Our results clearly show that this simple criterion is not adequate for screening unduly contaminated samples. The difference in amino acid concentration in contaminated and uncontaminated samples was as much as one order of magnitude for aspartate, glutamate, glycine and taurine even though all samples appeared macroscopically uncontaminated. Variations in the concentrations of different amino acids may be partly due to reported normal variations in the plasmaCSF gradient (e.g. ref. 4). However, the correlation between the amino acid concentration and the number of erythrocytes in the CSF is higher than could be expected simply on the basis of plasma-CSF gradients. Further factors which might explain our findings could be the high concentration of certain amino acids in blood cells [3, 5] and blood enzymes causing amino acid changes in the CSF. From our results we conclude that the contribution of non-visible contamination by blood to measures of CSF constituents should be assessed at least for substances known to have high blood-CSF gradients. We wish to thank H. Zettlmeissl for the facility to carry out the HPLC measurements. 1 Kornhuber, J., Kim, J.S. and Kornhuber, M.E., CSF sampling techniques. In J.H. Hingtgen, D.H., Hellhammer and G. Huppmann (Eds.), Advanced Methods in Psychobiology, Hogrefe, Toronto, in press. 2 Patten, B.M., How.much blood makes the cerebrospinal fluid bloody?, JAMA, 206 (1968) 378. 3 Perry, T.L. and Hansen, S., Technical pitfalls leading to errors in the quantitation of plasma amino acids, Clin. Chim. Acta, 25 (1969) 53-58. 4 Perry, T.L., Hansen, S. and Kennedy, J., CSF amino acids and plasma-CSF amino acid ratios in adults, J. Neurochem., 24 (1975) 587-589. 5 Soupart, P., Free amino acids of blood and urine in the human. In J.T. Holden (Ed.), Amino Acid Pools, Elsevier, Amsterdam, 1962, pp. 220-262.
215 6 Ylitalo, P., Heikkinen, E.R. and Myllylfi V.V., Evaluation of successive collection of cisternal cerebrospinal fluid in rals, rabbits, and cats, Exp. Neurol., 50 (1976) 330-336. 7 Zettlmeissl, H., Blome, J. and Kornhuber, H.H., A sensitive, fast, durable, highly sensitive method for the determination of amino acids and biogcnic amines in the cerebrospinal fluid and other body fluids and tissues, Arch. hal. Biol., in press.