Clin Biochern, Vol. 23, pp. 237-240, 1990 Printed in Canada. All rights reserved.
0009-9120/90 $3.00 + .00
Copyright © 1990 The Canadian Society of Clinical Chemist~.
The Effects of Changes in Plasma Amino Acid Concentrations on Erythrocyte Amino Acid Content ADRIEN SCHAEFER, FRANCOIS PIQUARD, and PASCAL HABEREY Groupe de Recherche en Nutrition Foetale, Universite Louis Pasteur, Faculte de Medecine, F-67085 Strasbourg Cedex, France In order to determine the effects of large variations in plasma amino acid concentrations upon human erythrocyte amino acid content, the plasma concentration of blood samples was enhanced (x 3.8) by adding amino acids or decreased (x 0.49) by plasma dilution. Before and after incubation (30 s at 37 °C), the erythrocyte contents were calculated from whole blood and plasma amino acid concentrations. Large and rapid plasma concentration variations led to significant erythrocyte changes in 11 amino acids. THR, CIT, (~AB, VAL, MET, ILE, LEU, TYR, PHE, TRP, and ARG. Relationships between erythrocyte and plasma concentrations were determined for these amino acids. These observations were examined in the light of the role played by erythrocytes in blood amino acid transport.
KEY WORDS: amino acids; plasma; erythrocytes; human. he determination of free amino acids in biological fluids has become routine in diagnosing metabolic disorders. Amino acids are usually determined in plasma rather than in whole blood. The fact that there are differences between some plasma and erythrocyte amino acid concentrations (1,2) and that erythrocytes participate in inter-organ transport of amino acids (1,3-7) has raised the question of the value of using these two determinations clinically. It has become necessary, therefore, to study whether relationships between erythrocytes and plasma exist and whether plasma variations in amino acid concentrations influence erythrocyte content. Inter-organ transport studies in h u m a n (1,4,5) and animal species (3,6,7) provide information concerning the role of erythrocytes in amino acid transport. Amino acid exchanges were measured by arteriovenous differences in whole blood, in plasma or in both. Discrepancies in the respective roles of plasma and red blood cells in transport of certain amino acids were often found, possibly due to species variations and to differences in experimental techniques.
T
Correspondence: A. Schaefer, I n s t i t u i ~ e Physiologie, Facult~ de M~decine, F-67085 Strasbourg Cedex, France.
Manuscript received April 25, 1989; revised October 3, 1989; Accepted October 12, 1989. Abbreviations: TAU = taurine; aAB = ~-aminobutyrate; CIT = citrulline. All other abbreviations for amino acids follow standard IUB recommendations. CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990
These discrepancies could also be explained by the fact that arterio-venous differences determined in vivo are so small (8), especially in whole blood, that they often remain undetected. The aim of the present experiments was to study, in vitro, the effects of rapid changes in plasma amino acid concentrations upon erythrocyte concentrations in human blood. The release and removal of amino acids in organ tissues were simulated by adding amino acids to the blood and by decreasing plasma concentration through dilution. The large simulated arterio-venous differences, which appear in this procedure, can be easily measured. Methods Amino acid analyses were performed by ion exchange chromatography using the LIQUIMAT II (Roche Kontron). The method using a single column of DC 6 resin (Durrum) and lithium Pico-buffer System IV (Durrum) and fluorimetric detection has already been described in a previous study (9). This method does not permit determination of proline and hydroxyproline concentrations. The samples were deproteinized by adding 100 ~L of a 2 mol/L sulfosalicylic acid solution to either 1 mL of plasma or 1 mL of hemolysed blood. After shaking, the precipitate was centrifuged for 20 min at 18,000 x g. Exactly 100 ~L of the supernatant was diluted with 300 ILL of a citrate buffer containing aminoadipic acid as internal standard for amino acid analysis. The composition of this buffer was: lithium citrate 4H20 , 0.087 mol/L, aminoadipic acid 66.6 ~mol/L, thioglycol 0.024 mol/L, octanoic acid 0.6 mmol/L dissolved in double distilled water and adjusted with concentrated hydrochloric acid to a final pH of 2.2. For whole blood samples, to accomplish complete hemolysis before deproteinization, 1 mL of blood was diluted with 1.5 mL of distilled water and then frozen twice in liquid nitrogen. Blood samples (approximately 20 mL) were obtained by venepuncture from the antecubital vein of five healthy male volunteers (aged 19-53), using a previously heparinized syringe. The blood was kept on ice and divided into three aliquots. The first aliquot (NORM) was used to determine normal plasma and whole blood amino acid concen237
SCHAEFER, PIQUARD, AND HABEREY TABLE 1 Concentrations of Free Amino Acids in Plasma, Whole Blood and Erythrocytes Before (NORM), After Enhancing (CONC) or Decreasing (DIL) Plasma Amino Acids Plasma NORM Taurine 60 ± 8 Aspartate 7 ± 1 Threonine 149 ± 45 Serine 114 ± 45 Asparagine 60 ± 12 Glutamate 28 ± 9 Glutamine 1021 ± 225 Glycine 261 ± 102 Alanine 434 ± 96 Citrulline 40 ± 8 aAminobutyrate 24 ± 6 Valine 285 ± 82 Cystine 81 ± 15 Methionine 32 ± 6 Isoleucine 74 ± 16 Leucine 156 ± 38 Tyrosine 76 ± 15 Phenylalanine 63 ± 17 Ornithine 94 ± 35 Lysine 208 ± 47 Histidine 99 ± 7 Tryptophan 67 ± 8 Arginine 101 ± 14 Total
Whole Blood NORM 280 961 155 157 101 197 886 362 421 42 22 255 34 79 148 85 84 172 193 97 51 68
± ± ± ± ± ± ± ± ± ± ± ± . ± ± ± ± ± ± ± ± ± ±
NORM
Erythrocytes CONC
101" 539 ± 216 437 ± 200 158" 2081 ± 343 1916 ± 425 35 163 ± 30 199 ± 46 41 207 ± 47 214 ± 80 32* 150 ± 58 159 ± 68 50* 396 ± 106 396 ± 162 188 813 ± 181 884 ± 213 103 479 ± 112 466 ± 82 81 406 ± 82 464 ± 84 11 44 ± 14 64 ± 13 6 21 ± 6 36 ± 12 77 219 ± 72 414 ± 79 . . . 6 36 ± 8 84 ± 20 20 85 ± 25 147 ± 30 36 139 ± 33 274 ± 31 20 96 ± 27 126 ± 27 23 108 ± 37 146 ± 35 33* 265 ± 50 249 ± 189 50 175 ± 60 173 ± 85 20 95 ± 36 120 ± 39 8* 32 ± 21 99 ± 50 17" 29 ± 24 73 ± 18
3331 ± 874 4854 ± 856
DIL 480 1952 137 178 135 328 714 432 367 44 22 151 25 67 92 80 64 171 153 96 20 52
± 191 ± 373 -+ 27 ¢ ± 40 ± 46 ± 108 ± 131 ± 78 ± 76 ± 21 a ± 10a¢ ± 55 ~¢ ± ± ± ±
4 ~b¢ 18a¢ 25 ~b¢ 12¢
± 2 7 abe
± ± ± ± ±
68 35 19 16a¢ 12~
6643 ± 989 7766 ± 1896 5716 ± 1016
Values are means ± SD (n = 5). Erythrocyte values were calculated from plasma and whole blood values (see Methods). *Significant differences (p < 0.05) between NORM Plasma and NORM Whole Blood. a'b'¢Comparisons within the calculated erythrocyte concentrations p < 0.05: aNORM compared to CONC, bNORM compared to DIL, ¢CONC compared to DIL. trations. The second (DIL) a n d the t h i r d (CONC) aliquots were c e n t r i f u g e d a t 1500 × g for I rain, a n d t h e n 500 ~LL of p l a s m a per 1 m L of blood was removed. A n equal v o l u m e of e i t h e r R i n g e r solution (aliquot 2, DIL) or R i n g e r solution c o n t a i n i n g a b o u t four t i m e s the n o r m a l p l a s m a a m i n o acid c o n c e n t r a tions (aliquot 3, CONC) was t h e n added. T h e final p H was adjusted to 7.35 b y a d d i n g s o d i u m hydroxide. The two m i x t u r e s were t h e n g e n t l y s h a k e n a n d placed in a w a t e r b a t h (37 °C) for exactly 30 s. T h e y were t h e n p u t on ice once again. The t h r e e blood p r e p a r a t i o n s , w h i c h correspond respectively to n o r m a l , diluted, a n d c o n c e n t r a t e d blood, were a n a l y z e d for p l a s m a (P) a n d whole blood ( W B ) a m i n o acid concentrations. The e r y t h r o c y t e c o n c e n t r a t i o n s (E) were calculated from p l a s m a a n d whole blood c o n c e n t r a t i o n s , u s i n g t h e following formula: (E) = ( W B ) - (1 - Hct).(P) / Hct. H c t is t h e p a c k e d cell volume. Differences in w a t e r
c o n t e n t o f e r y t h r o c y t e s c o m p a r e d to p l a s m a were not t a k e n into account. 238
S t a t i s t i c a l a n a l y s i s was p e r f o r m e d u s i n g the nonp a r a m e t r i c M a n n - W h i t n e y test for single comparisons a n d t h e n o n p a r a m e t r i c K r u s k a l l - W a l l i s r a n g e test for m u l t i p l e c o m p a r i s o n s (10). W h e n c o r r e l a t i o n a n a l y s i s w a s used, c a l c u l a t i o n of l i n e a r r e g r e s s i o n s was r e s t r i c t e d to p a i r e d d a t a w i t h s i g n i f i c a n t correlation coefficients (p < 0.05).
Results Table 1 s u m m a r i z e s t h e m e a n a n d SD of p l a s m a , whole blood a n d e r y t h r o c y t e c o n c e n t r a t i o n s of a m i n o acids in the n o r m a l (NORM) blood a n d e r y t h r o c y t e c o n c e n t r a t i o n s in diluted (DIL) a n d c o n c e n t r a t e d (CONC) blood. Studies on t h e d i s a p p e a r a n c e of cystine in whole blood s a m p l e s (11) h a v e s h o w n t h a t this w a s due to its disulphide b i n d i n g to t h e s u l p h y dryl g r o u p s of p l a s m a proteins d u r i n g hemolysis. The r a p i d d e p r o t e i n i z a t i o n a f t e r c e n t r i f u g a t i o n avoided this c h a n g e in p l a s m a samples. T h e m e a n n o r m a l p l a s m a c o n c e n t r a t i o n of G L N w a s s o m e w h a t h i g h e r t h a n p r e v i o u s l y r e p o r t e d values (2,4,5) a n d exceeded t h e n o r m a l r a n g e u s u a l l y g i v e n in clinical reference tables. This m a y be due to CLINICAL BIOCHEMISTRY,VOLUME 23, JUNE 1990
PLASMA AND ERYTHROCYTE
TABLE2 Correlation Coefficients (r) and Regression Coefficients (R) Between Erythrocyte and Plasma Amino Acid Concentrations N = 15 Taurine Aspartate Threonine Serine Asparagine Glutamate Glutamine Glycine Alanine Citrulline ~Aminobutyrate Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Tryptophan Arginine
r 0.057 0.021 0.689 0.360 0.342 0.301 0.369 0.285 0.467 0.732 0.725 0.961 0.907 0.830 0.949 0.666 0.665 0.219 0.220 0.330 0.853 0.514
Significance NS NS 0.01 NS NS NS NS NS NS 0.01 0.01 0.001 0.001 0.001 0.001 0.01 0.01 NS NS NS 0.001 0.05
R -0.12 ------0.33 0.22 0.38 0.24 0.39 0.50 0.26 0.50 ---0.52 0.10
the methodology: the immediate processing of blood samples avoided the rapid deamination of GLN to GLU (9). Secondly, this mean value is enhanced by an unexplained high concentration observed in one of the five subjects (1300 ~Lmol/L). In NORM blood, plasma and whole blood amino acid concentrations were significantly different for seven amino acids. TAU, ASP, ASN, GLU, and ORN were higher in whole blood t h a n in plasma whereas TRP and ARG were lower. The calculated erythrocyte concentrations of amino acids from NORM, CONC, and DIL blood show t h a t the increase or decrease in plasma concentrations did not change the erythrocyte content for all amino acids. When changes in erythrocytes occurred, they were in the same direction as those in the plasma. Considerable enhancement of all plasma amino acids (mean x 3.8) produced a significant increase in the erythrocyte concentrations of only nine (CIT, ~AB, VAL, MET, ILE, LEU, PHE, TRP, and ARG), whereas decreases (mean x 0.49) produced a significant decrease in three (MET, LEU, and PHE). For two other amino acids (THR and TYR) changes appeared only by comparing erythrocyte concentrations of CONC and DIL blood. Thus variations of plasma concentrations led to statistically significant changes in the red blood cells in 11 amino acids (THR, CIT, ~AB, VAL, MET, ILE, LEU, TYR, PHE, TRP, and ARG). CLINICALBIOCHEMISTRY,VOLUME 23, JUNE 1990
A M I N O ACIDS
Table 2 outlines the relationships between the plasma concentrations of NORM, CONC, and DIL blood and the corresponding erythrocyte values. The correlation coefficients were significant for the same 11 amino acids mentioned above (THR, CIT, aAB, VAL, MET, ILE, LEU, TYR, PHE, TRP, and ARG). These data show t h a t the erythrocyte concentrations of these 11 amino acids are dependent on the plasma concentrations and t h a t exchanges take place within 30 s of contact between plasma and erythrocytes. The slopes of the regression lines were always below 1, varying from 0.12 (THR) to 0.52 (TRP). The best correlation coefficients were found for VAL, LEU, and MET. Considering the total of all determined amino acids, a 266% enhancement of total plasma concentration resulted in an increase of 17% in the erythrocyte concentration. A 49% diminution was followed by a decrease of 12% in the erythrocyte content after 30 s of incubation. Analysis of the original erythrocyte concentrations showed t h a t those of the 11 amino acids carried from plasma to red cells were lower t h a n those of the other amino acids. This suggests t h a t the changes in erythrocyte concentrations depend upon their original concentrations. To validate this hypothesis, we looked for correlations between the original erythrocyte concentrations and observed changes taking into account the individual values of all the amino acids. After enhancement of plasma amino acids, a significant correlation was found (r = 0.421, p < 0.0001). The calculated regression coefficient (R = -0.10) indicated t h a t the changes of erythrocyte amino acids concentrations (CONC-NORM differences) were inversely proportional to the original amino acid concentrations (NORM). The amino acids with high original concentrations showed the smallest changes. By contrast, after decrease of plasma amino acids, a significant and positive relationship was obtained (r = 0.536, p < 0.0001, R = 0.06). The changes (NORM-DIL differences) were directly proportional to the original concentrations.
Discussion The plasma, whole blood and erythrocyte concentrations of amino acids presented in Table 1 for normal h u m a n blood (NORM) were compatible with previously reported results (1,2). The large plasma changes, which were artificially induced, allowed optimization of the phenomenon of plasma -- erythrocyte exchanges of amino acids to a much larger extent t h a n would have occurred in vivo. Thus, the erythrocyte concentrations of 11 amino acids correlated with those of the plasma in this study. During the short time of contact, only some amino acids crossed the erythrocyte membrane from the highest to the lowest concentration. Other amino acids were retained in the red blood cells and their intracellular concentrations were independent 239
SCHAEFER, PIQUARD, AND HABEREY
of those of the plasma. These observations are in accordance with the findings of Hagenfeldt and Arvidsson (2) who showed t h a t only certain amino acids, i.e., MET, VAL, ILE, LEU, TYR, and PHE were partially washed out of erythrocytes, whereas the other amino acids remained inside the cell in spite of being at a higher concentration t h a n in the incubation medium. The inversely proportional correlation found between the changes and the original concentrations in the erythrocytes after plasma amino acid enhancement suggests t h a t a maximal concentration for each amino acid which cannot be exceeded may exist in the erythrocyte. The existence of a saturation concentration in the red cells could explain why small changes are observed for some amino acids in which this concentration may be reached or approached. In contrast, when this saturation is not reached, large changes in amino acid concentration are observed. The directly proportional relationship obtained between the changes and the original concentrations in the erythrocytes after plasma amino acid decrease further supports this hypothesis. When considering the results in this in vitro study with those obtained in inter-organ amino acid transport studies in vivo, it must be kept in mind t h a t the shifts of plasma amino acid concentrations, as well as the time of contact between the red blood cells and the plasma, were maximized in our study. In spite of both being much greater t h a n when blood flows through an organ, the participation of erythrocytes in the transport of the metabolically active amino acids GLU and ALA in h u m a n blood (1,4,5) was not obvious here. On the contrary, in this study, it was mainly the essential amino acids (with the exception of lysine) which were transported by the erythrocytes. LEU appears to be an amino acid which easily crossed the cell membranes of red blood cells when plasma amino acid concentrations were modified, as shown by the highly significant correlation coefficient observed between its plasma and erythrocyte concentrations. This observation supports the regulating effect of LEU on protein synthesis in muscle and heart tissues (12,13). It must be noted t h a t our experiments do not take into account a possible direct exchange between erythrocytes and tissues which has been suggested by Elwyn et al. (3). The existence of such a direct exchange remains to be proven. Nevertheless, erythrocyte transport remains small when compared with the plasma transport. Other studies performed in vitro (14) and in vivo (5) have already shown t h a t the equilibration time of plasma and erythrocyte amino acid concentrations is rather long. Therefore, it seems improbable t h a t in vivo important amino acid
240
exchanges between organ tissues and erythrocytes using plasma as an intermediate step can occur during the time of contact. In our study, rapid exchanges between plasma and erythrocytes were rather small and concerned only 11 amino acids. This supports the use of plasma determinations to study inter-organ amino acid exchange or for diagnosing metabolic disorders. Amino acid imbalance can therefore be detected in plasma as well as in whole blood. References 1. Felig P, Wahren J, R~if L. Evidence of inter-organ amino-acid transport by blood cells in humans. Proc Natl Acad Sci USA 1973; 70: 1775-9. 2. Hagenfeldt L, Arvidsson A. The distribution of amino acids between plasma and erythrocytes. Clin Chim Acta 1980; 100: 133-41. 3. Elwyn DH, Launder WJ, Parikh HC, Wise EM Jr. Role of plasma and erythrocytes in inter-organ transport of amino acids in dogs. A m J Physiol 1972; 222: 1333-42. 4. Aoki TT, Brennan MF, Mtiller WA, Moore FD, Cahill GF Jr. Effect of insulin on muscle glutamate uptake. Whole blood versus plasma glutamate analysis. J Clin Invest 1972; 51: 2889-94. 5. Aoki TT, Mtiller WA, Brennan MF, Cahill GF Jr. Blood cell and plasma amino acid levels across forearm muscle during a protein meal. Diabetes 1973; 22: 768-75. 6. Drewes LR, Conway WP, Gilboe DD. Net amino acid transport between plasma and erythrocytes and perfused dog brain. A m J Physiol 1977; 233: E320-E325. 7. Heitmann RN, Bergman EN. Transport of amino acids in whole blood and plasma of sheep. A m J Physiol 1980; 239: E242-E247. 8. Pitts RF, de Haas J, Klein J. Relation of renal amino and amide nitrogen extraction to ammonia production. A m J Physiol 1963; 204: 187-91. 9. Schaefer A, Piquard F, Haberey P. Plasma aminoacids analysis: effects of delayed samples preparation and of storage. Clin Chim Acta 1987; 164: 163-9. 10. Siegel S. Nonparametric statistics for the behavioural sciences. New York: McGraw-Hill Book Company, 1956. 11. Perry TL, Hansen S. Technical pitfalls leading to errors in the quantitation of plasma amino acid. Clin Chim Acta 1969; 25: 53-8. 12. Buse MG, Weigand DA. Studies concerning the specificity of the effect of leucine on the turnover of proteins in muscles of control and diabetic rats. Biochim Biophys Acta 1977; 475: 81-9. 13. Chua B, Siehl DL, Morgan HE. Effect of leucine and metabolites of branched chain amino acids on protein turnover in heart. J Biol Chem 1979; 254: 8358-62. 14. Winter CG, Christensen HN. Migration of amino acids across the membrane of the human erythrocyte. J Biol Chem 1964; 239: 872-8.
CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990
0009-9120/90 $3.00 + .00
Copyright © 1990 The Canadian Society of Clinical Chemist~.
The Effects of Changes in Plasma Amino Acid Concentrations on Erythrocyte Amino Acid Content ADRIEN SCHAEFER, FRANCOIS PIQUARD, and PASCAL HABEREY Groupe de Recherche en Nutrition Foetale, Universite Louis Pasteur, Faculte de Medecine, F-67085 Strasbourg Cedex, France In order to determine the effects of large variations in plasma amino acid concentrations upon human erythrocyte amino acid content, the plasma concentration of blood samples was enhanced (x 3.8) by adding amino acids or decreased (x 0.49) by plasma dilution. Before and after incubation (30 s at 37 °C), the erythrocyte contents were calculated from whole blood and plasma amino acid concentrations. Large and rapid plasma concentration variations led to significant erythrocyte changes in 11 amino acids. THR, CIT, (~AB, VAL, MET, ILE, LEU, TYR, PHE, TRP, and ARG. Relationships between erythrocyte and plasma concentrations were determined for these amino acids. These observations were examined in the light of the role played by erythrocytes in blood amino acid transport.
KEY WORDS: amino acids; plasma; erythrocytes; human. he determination of free amino acids in biological fluids has become routine in diagnosing metabolic disorders. Amino acids are usually determined in plasma rather than in whole blood. The fact that there are differences between some plasma and erythrocyte amino acid concentrations (1,2) and that erythrocytes participate in inter-organ transport of amino acids (1,3-7) has raised the question of the value of using these two determinations clinically. It has become necessary, therefore, to study whether relationships between erythrocytes and plasma exist and whether plasma variations in amino acid concentrations influence erythrocyte content. Inter-organ transport studies in h u m a n (1,4,5) and animal species (3,6,7) provide information concerning the role of erythrocytes in amino acid transport. Amino acid exchanges were measured by arteriovenous differences in whole blood, in plasma or in both. Discrepancies in the respective roles of plasma and red blood cells in transport of certain amino acids were often found, possibly due to species variations and to differences in experimental techniques.
T
Correspondence: A. Schaefer, I n s t i t u i ~ e Physiologie, Facult~ de M~decine, F-67085 Strasbourg Cedex, France.
Manuscript received April 25, 1989; revised October 3, 1989; Accepted October 12, 1989. Abbreviations: TAU = taurine; aAB = ~-aminobutyrate; CIT = citrulline. All other abbreviations for amino acids follow standard IUB recommendations. CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990
These discrepancies could also be explained by the fact that arterio-venous differences determined in vivo are so small (8), especially in whole blood, that they often remain undetected. The aim of the present experiments was to study, in vitro, the effects of rapid changes in plasma amino acid concentrations upon erythrocyte concentrations in human blood. The release and removal of amino acids in organ tissues were simulated by adding amino acids to the blood and by decreasing plasma concentration through dilution. The large simulated arterio-venous differences, which appear in this procedure, can be easily measured. Methods Amino acid analyses were performed by ion exchange chromatography using the LIQUIMAT II (Roche Kontron). The method using a single column of DC 6 resin (Durrum) and lithium Pico-buffer System IV (Durrum) and fluorimetric detection has already been described in a previous study (9). This method does not permit determination of proline and hydroxyproline concentrations. The samples were deproteinized by adding 100 ~L of a 2 mol/L sulfosalicylic acid solution to either 1 mL of plasma or 1 mL of hemolysed blood. After shaking, the precipitate was centrifuged for 20 min at 18,000 x g. Exactly 100 ~L of the supernatant was diluted with 300 ILL of a citrate buffer containing aminoadipic acid as internal standard for amino acid analysis. The composition of this buffer was: lithium citrate 4H20 , 0.087 mol/L, aminoadipic acid 66.6 ~mol/L, thioglycol 0.024 mol/L, octanoic acid 0.6 mmol/L dissolved in double distilled water and adjusted with concentrated hydrochloric acid to a final pH of 2.2. For whole blood samples, to accomplish complete hemolysis before deproteinization, 1 mL of blood was diluted with 1.5 mL of distilled water and then frozen twice in liquid nitrogen. Blood samples (approximately 20 mL) were obtained by venepuncture from the antecubital vein of five healthy male volunteers (aged 19-53), using a previously heparinized syringe. The blood was kept on ice and divided into three aliquots. The first aliquot (NORM) was used to determine normal plasma and whole blood amino acid concen237
SCHAEFER, PIQUARD, AND HABEREY TABLE 1 Concentrations of Free Amino Acids in Plasma, Whole Blood and Erythrocytes Before (NORM), After Enhancing (CONC) or Decreasing (DIL) Plasma Amino Acids Plasma NORM Taurine 60 ± 8 Aspartate 7 ± 1 Threonine 149 ± 45 Serine 114 ± 45 Asparagine 60 ± 12 Glutamate 28 ± 9 Glutamine 1021 ± 225 Glycine 261 ± 102 Alanine 434 ± 96 Citrulline 40 ± 8 aAminobutyrate 24 ± 6 Valine 285 ± 82 Cystine 81 ± 15 Methionine 32 ± 6 Isoleucine 74 ± 16 Leucine 156 ± 38 Tyrosine 76 ± 15 Phenylalanine 63 ± 17 Ornithine 94 ± 35 Lysine 208 ± 47 Histidine 99 ± 7 Tryptophan 67 ± 8 Arginine 101 ± 14 Total
Whole Blood NORM 280 961 155 157 101 197 886 362 421 42 22 255 34 79 148 85 84 172 193 97 51 68
± ± ± ± ± ± ± ± ± ± ± ± . ± ± ± ± ± ± ± ± ± ±
NORM
Erythrocytes CONC
101" 539 ± 216 437 ± 200 158" 2081 ± 343 1916 ± 425 35 163 ± 30 199 ± 46 41 207 ± 47 214 ± 80 32* 150 ± 58 159 ± 68 50* 396 ± 106 396 ± 162 188 813 ± 181 884 ± 213 103 479 ± 112 466 ± 82 81 406 ± 82 464 ± 84 11 44 ± 14 64 ± 13 6 21 ± 6 36 ± 12 77 219 ± 72 414 ± 79 . . . 6 36 ± 8 84 ± 20 20 85 ± 25 147 ± 30 36 139 ± 33 274 ± 31 20 96 ± 27 126 ± 27 23 108 ± 37 146 ± 35 33* 265 ± 50 249 ± 189 50 175 ± 60 173 ± 85 20 95 ± 36 120 ± 39 8* 32 ± 21 99 ± 50 17" 29 ± 24 73 ± 18
3331 ± 874 4854 ± 856
DIL 480 1952 137 178 135 328 714 432 367 44 22 151 25 67 92 80 64 171 153 96 20 52
± 191 ± 373 -+ 27 ¢ ± 40 ± 46 ± 108 ± 131 ± 78 ± 76 ± 21 a ± 10a¢ ± 55 ~¢ ± ± ± ±
4 ~b¢ 18a¢ 25 ~b¢ 12¢
± 2 7 abe
± ± ± ± ±
68 35 19 16a¢ 12~
6643 ± 989 7766 ± 1896 5716 ± 1016
Values are means ± SD (n = 5). Erythrocyte values were calculated from plasma and whole blood values (see Methods). *Significant differences (p < 0.05) between NORM Plasma and NORM Whole Blood. a'b'¢Comparisons within the calculated erythrocyte concentrations p < 0.05: aNORM compared to CONC, bNORM compared to DIL, ¢CONC compared to DIL. trations. The second (DIL) a n d the t h i r d (CONC) aliquots were c e n t r i f u g e d a t 1500 × g for I rain, a n d t h e n 500 ~LL of p l a s m a per 1 m L of blood was removed. A n equal v o l u m e of e i t h e r R i n g e r solution (aliquot 2, DIL) or R i n g e r solution c o n t a i n i n g a b o u t four t i m e s the n o r m a l p l a s m a a m i n o acid c o n c e n t r a tions (aliquot 3, CONC) was t h e n added. T h e final p H was adjusted to 7.35 b y a d d i n g s o d i u m hydroxide. The two m i x t u r e s were t h e n g e n t l y s h a k e n a n d placed in a w a t e r b a t h (37 °C) for exactly 30 s. T h e y were t h e n p u t on ice once again. The t h r e e blood p r e p a r a t i o n s , w h i c h correspond respectively to n o r m a l , diluted, a n d c o n c e n t r a t e d blood, were a n a l y z e d for p l a s m a (P) a n d whole blood ( W B ) a m i n o acid concentrations. The e r y t h r o c y t e c o n c e n t r a t i o n s (E) were calculated from p l a s m a a n d whole blood c o n c e n t r a t i o n s , u s i n g t h e following formula: (E) = ( W B ) - (1 - Hct).(P) / Hct. H c t is t h e p a c k e d cell volume. Differences in w a t e r
c o n t e n t o f e r y t h r o c y t e s c o m p a r e d to p l a s m a were not t a k e n into account. 238
S t a t i s t i c a l a n a l y s i s was p e r f o r m e d u s i n g the nonp a r a m e t r i c M a n n - W h i t n e y test for single comparisons a n d t h e n o n p a r a m e t r i c K r u s k a l l - W a l l i s r a n g e test for m u l t i p l e c o m p a r i s o n s (10). W h e n c o r r e l a t i o n a n a l y s i s w a s used, c a l c u l a t i o n of l i n e a r r e g r e s s i o n s was r e s t r i c t e d to p a i r e d d a t a w i t h s i g n i f i c a n t correlation coefficients (p < 0.05).
Results Table 1 s u m m a r i z e s t h e m e a n a n d SD of p l a s m a , whole blood a n d e r y t h r o c y t e c o n c e n t r a t i o n s of a m i n o acids in the n o r m a l (NORM) blood a n d e r y t h r o c y t e c o n c e n t r a t i o n s in diluted (DIL) a n d c o n c e n t r a t e d (CONC) blood. Studies on t h e d i s a p p e a r a n c e of cystine in whole blood s a m p l e s (11) h a v e s h o w n t h a t this w a s due to its disulphide b i n d i n g to t h e s u l p h y dryl g r o u p s of p l a s m a proteins d u r i n g hemolysis. The r a p i d d e p r o t e i n i z a t i o n a f t e r c e n t r i f u g a t i o n avoided this c h a n g e in p l a s m a samples. T h e m e a n n o r m a l p l a s m a c o n c e n t r a t i o n of G L N w a s s o m e w h a t h i g h e r t h a n p r e v i o u s l y r e p o r t e d values (2,4,5) a n d exceeded t h e n o r m a l r a n g e u s u a l l y g i v e n in clinical reference tables. This m a y be due to CLINICAL BIOCHEMISTRY,VOLUME 23, JUNE 1990
PLASMA AND ERYTHROCYTE
TABLE2 Correlation Coefficients (r) and Regression Coefficients (R) Between Erythrocyte and Plasma Amino Acid Concentrations N = 15 Taurine Aspartate Threonine Serine Asparagine Glutamate Glutamine Glycine Alanine Citrulline ~Aminobutyrate Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Tryptophan Arginine
r 0.057 0.021 0.689 0.360 0.342 0.301 0.369 0.285 0.467 0.732 0.725 0.961 0.907 0.830 0.949 0.666 0.665 0.219 0.220 0.330 0.853 0.514
Significance NS NS 0.01 NS NS NS NS NS NS 0.01 0.01 0.001 0.001 0.001 0.001 0.01 0.01 NS NS NS 0.001 0.05
R -0.12 ------0.33 0.22 0.38 0.24 0.39 0.50 0.26 0.50 ---0.52 0.10
the methodology: the immediate processing of blood samples avoided the rapid deamination of GLN to GLU (9). Secondly, this mean value is enhanced by an unexplained high concentration observed in one of the five subjects (1300 ~Lmol/L). In NORM blood, plasma and whole blood amino acid concentrations were significantly different for seven amino acids. TAU, ASP, ASN, GLU, and ORN were higher in whole blood t h a n in plasma whereas TRP and ARG were lower. The calculated erythrocyte concentrations of amino acids from NORM, CONC, and DIL blood show t h a t the increase or decrease in plasma concentrations did not change the erythrocyte content for all amino acids. When changes in erythrocytes occurred, they were in the same direction as those in the plasma. Considerable enhancement of all plasma amino acids (mean x 3.8) produced a significant increase in the erythrocyte concentrations of only nine (CIT, ~AB, VAL, MET, ILE, LEU, PHE, TRP, and ARG), whereas decreases (mean x 0.49) produced a significant decrease in three (MET, LEU, and PHE). For two other amino acids (THR and TYR) changes appeared only by comparing erythrocyte concentrations of CONC and DIL blood. Thus variations of plasma concentrations led to statistically significant changes in the red blood cells in 11 amino acids (THR, CIT, ~AB, VAL, MET, ILE, LEU, TYR, PHE, TRP, and ARG). CLINICALBIOCHEMISTRY,VOLUME 23, JUNE 1990
A M I N O ACIDS
Table 2 outlines the relationships between the plasma concentrations of NORM, CONC, and DIL blood and the corresponding erythrocyte values. The correlation coefficients were significant for the same 11 amino acids mentioned above (THR, CIT, aAB, VAL, MET, ILE, LEU, TYR, PHE, TRP, and ARG). These data show t h a t the erythrocyte concentrations of these 11 amino acids are dependent on the plasma concentrations and t h a t exchanges take place within 30 s of contact between plasma and erythrocytes. The slopes of the regression lines were always below 1, varying from 0.12 (THR) to 0.52 (TRP). The best correlation coefficients were found for VAL, LEU, and MET. Considering the total of all determined amino acids, a 266% enhancement of total plasma concentration resulted in an increase of 17% in the erythrocyte concentration. A 49% diminution was followed by a decrease of 12% in the erythrocyte content after 30 s of incubation. Analysis of the original erythrocyte concentrations showed t h a t those of the 11 amino acids carried from plasma to red cells were lower t h a n those of the other amino acids. This suggests t h a t the changes in erythrocyte concentrations depend upon their original concentrations. To validate this hypothesis, we looked for correlations between the original erythrocyte concentrations and observed changes taking into account the individual values of all the amino acids. After enhancement of plasma amino acids, a significant correlation was found (r = 0.421, p < 0.0001). The calculated regression coefficient (R = -0.10) indicated t h a t the changes of erythrocyte amino acids concentrations (CONC-NORM differences) were inversely proportional to the original amino acid concentrations (NORM). The amino acids with high original concentrations showed the smallest changes. By contrast, after decrease of plasma amino acids, a significant and positive relationship was obtained (r = 0.536, p < 0.0001, R = 0.06). The changes (NORM-DIL differences) were directly proportional to the original concentrations.
Discussion The plasma, whole blood and erythrocyte concentrations of amino acids presented in Table 1 for normal h u m a n blood (NORM) were compatible with previously reported results (1,2). The large plasma changes, which were artificially induced, allowed optimization of the phenomenon of plasma -- erythrocyte exchanges of amino acids to a much larger extent t h a n would have occurred in vivo. Thus, the erythrocyte concentrations of 11 amino acids correlated with those of the plasma in this study. During the short time of contact, only some amino acids crossed the erythrocyte membrane from the highest to the lowest concentration. Other amino acids were retained in the red blood cells and their intracellular concentrations were independent 239
SCHAEFER, PIQUARD, AND HABEREY
of those of the plasma. These observations are in accordance with the findings of Hagenfeldt and Arvidsson (2) who showed t h a t only certain amino acids, i.e., MET, VAL, ILE, LEU, TYR, and PHE were partially washed out of erythrocytes, whereas the other amino acids remained inside the cell in spite of being at a higher concentration t h a n in the incubation medium. The inversely proportional correlation found between the changes and the original concentrations in the erythrocytes after plasma amino acid enhancement suggests t h a t a maximal concentration for each amino acid which cannot be exceeded may exist in the erythrocyte. The existence of a saturation concentration in the red cells could explain why small changes are observed for some amino acids in which this concentration may be reached or approached. In contrast, when this saturation is not reached, large changes in amino acid concentration are observed. The directly proportional relationship obtained between the changes and the original concentrations in the erythrocytes after plasma amino acid decrease further supports this hypothesis. When considering the results in this in vitro study with those obtained in inter-organ amino acid transport studies in vivo, it must be kept in mind t h a t the shifts of plasma amino acid concentrations, as well as the time of contact between the red blood cells and the plasma, were maximized in our study. In spite of both being much greater t h a n when blood flows through an organ, the participation of erythrocytes in the transport of the metabolically active amino acids GLU and ALA in h u m a n blood (1,4,5) was not obvious here. On the contrary, in this study, it was mainly the essential amino acids (with the exception of lysine) which were transported by the erythrocytes. LEU appears to be an amino acid which easily crossed the cell membranes of red blood cells when plasma amino acid concentrations were modified, as shown by the highly significant correlation coefficient observed between its plasma and erythrocyte concentrations. This observation supports the regulating effect of LEU on protein synthesis in muscle and heart tissues (12,13). It must be noted t h a t our experiments do not take into account a possible direct exchange between erythrocytes and tissues which has been suggested by Elwyn et al. (3). The existence of such a direct exchange remains to be proven. Nevertheless, erythrocyte transport remains small when compared with the plasma transport. Other studies performed in vitro (14) and in vivo (5) have already shown t h a t the equilibration time of plasma and erythrocyte amino acid concentrations is rather long. Therefore, it seems improbable t h a t in vivo important amino acid
240
exchanges between organ tissues and erythrocytes using plasma as an intermediate step can occur during the time of contact. In our study, rapid exchanges between plasma and erythrocytes were rather small and concerned only 11 amino acids. This supports the use of plasma determinations to study inter-organ amino acid exchange or for diagnosing metabolic disorders. Amino acid imbalance can therefore be detected in plasma as well as in whole blood. References 1. Felig P, Wahren J, R~if L. Evidence of inter-organ amino-acid transport by blood cells in humans. Proc Natl Acad Sci USA 1973; 70: 1775-9. 2. Hagenfeldt L, Arvidsson A. The distribution of amino acids between plasma and erythrocytes. Clin Chim Acta 1980; 100: 133-41. 3. Elwyn DH, Launder WJ, Parikh HC, Wise EM Jr. Role of plasma and erythrocytes in inter-organ transport of amino acids in dogs. A m J Physiol 1972; 222: 1333-42. 4. Aoki TT, Brennan MF, Mtiller WA, Moore FD, Cahill GF Jr. Effect of insulin on muscle glutamate uptake. Whole blood versus plasma glutamate analysis. J Clin Invest 1972; 51: 2889-94. 5. Aoki TT, Mtiller WA, Brennan MF, Cahill GF Jr. Blood cell and plasma amino acid levels across forearm muscle during a protein meal. Diabetes 1973; 22: 768-75. 6. Drewes LR, Conway WP, Gilboe DD. Net amino acid transport between plasma and erythrocytes and perfused dog brain. A m J Physiol 1977; 233: E320-E325. 7. Heitmann RN, Bergman EN. Transport of amino acids in whole blood and plasma of sheep. A m J Physiol 1980; 239: E242-E247. 8. Pitts RF, de Haas J, Klein J. Relation of renal amino and amide nitrogen extraction to ammonia production. A m J Physiol 1963; 204: 187-91. 9. Schaefer A, Piquard F, Haberey P. Plasma aminoacids analysis: effects of delayed samples preparation and of storage. Clin Chim Acta 1987; 164: 163-9. 10. Siegel S. Nonparametric statistics for the behavioural sciences. New York: McGraw-Hill Book Company, 1956. 11. Perry TL, Hansen S. Technical pitfalls leading to errors in the quantitation of plasma amino acid. Clin Chim Acta 1969; 25: 53-8. 12. Buse MG, Weigand DA. Studies concerning the specificity of the effect of leucine on the turnover of proteins in muscles of control and diabetic rats. Biochim Biophys Acta 1977; 475: 81-9. 13. Chua B, Siehl DL, Morgan HE. Effect of leucine and metabolites of branched chain amino acids on protein turnover in heart. J Biol Chem 1979; 254: 8358-62. 14. Winter CG, Christensen HN. Migration of amino acids across the membrane of the human erythrocyte. J Biol Chem 1964; 239: 872-8.
CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990
