Scand J Haematol(l975) 15, 178-186
Amino Acid Concentrations in Plasma and Erythrocytes in Aregeneratory and Haemolytic Anaemias MARTIN SEIP,M.D., ROLFLINDEMANN, M.D., PERGJESDAHL, Civ. Eng., & LEIVR.GJESSING, M.D. Pediatric Research Institute and Department of Pediatrics (Chief, M . Seip), Rikshospitalet, Oslo, and Dikernark Hospital, Asker, Norway
The concentrations of unbound amino acids in erythrocytes and in plasma from 7 normal individuals, 11 patients with various types of aregeneratory anaemia, and 4 patients with hereditary haemolytic anaemias were determined on a Technicon Amino Acid Analyzer (Perry et a1 1970). Most amino acids were normally found in higher concentrations in plasma than intracellularly. Cystine, methionine and tryptophan were almost exclusively present in plasma. Aspartic acid, however, was mainly found in erythrocytes, and glutathione only in erythrocytes. Glutamic acid and ornithine were more concentrated in the cells, while glycine and asparagine showed approximately the same concentrations in erythrocytes as in plasma. In the patients, plasma amino acids showed little deviations from normal, but in the erythrocytes there were striking changes. Erythrocyte glutamic acid concentrations were moderately to markedly elevated in all patients studied, and glycine concentrations in 13 out of 15 patients. In addition, the following amino acids were increased intracellularly in more than one patient: glutamine (8 patients), serine (7), asparagine (3,threonine (4), taurine (3), alanine (2), valine (2), ornithine (2), lysine (2), citrulline (2). Aspartic acid was decreased in erythrocytes from 4 patients with aregeneratory and 1 with haemolytic anaemia. Key words: amino acids - plasma
- erythrocytes - aregenerative anaemia
Accepted for publication June 24, 1975 Correspondence to: Prof. Martin Seip, Department of Pediatrics, Rikshospitalet, Oslo 1, Norway
Although much information is available concerning plasma concentrations of amino acids in health and disease, our knowledge of free amino acid content of red blood cells is sparse. A few authors have reported quantitative studies of amino acids in erythrocytes in normal individuals (McMenamy
et al 1960a/b, Soupart 1962, Levy & Barkin 1971), and in patients with various metabolic disorders (Barber & Spaeth 1969, Levy & Barkin 1971). Semiquantitative determinations of amino acids in red blood cells of healthy and diseased adults, and of foetal and maternal blood have also been
AMINO ACID CONCENTRATIONS IN ANAEMIAS
reported (Bj omesjo 1968ah). A few investigators have studied plasma and erythrocyte concentrations of unbound amino acids in various blood disorders, leukaemia (McMenamy et a1 1960b), haemolytic anaemias (Allen 1960), iron deficiency anaemia and haemolytic anaemias (Nicolaidou et a1 1965). Our interest in these problems was aroused during the study of two girls with congenital sideroblastic anaemia probably due to deltaaminolaevulinic acid (ALA) synthetase deficiency (Seip et a1 1971). Both patients showed very high levels of glycine and glutamic acid in their erythrocytes. The intention of this work was to study free amino acid Concentrations in erythrocytes of patients with aregeneratory and haemolytic anaemia, and to compare with the plasma concentrations. -03
;Fr,5\dA I
MATERIAL AND METHODS The patient material (Table I) consists of 11 patients with aregeneratory anaemia. The 4 patients with Diamond-Blackfan anaemia were on prednisone medication, while 1 patient with Fanconi anaemia and 1 with aplastic anaemia received anabolic steroids plus prednisone. In addition 4 patients with congenital haemolytic anaemia
were included. Most of the patients were children, however, 2 patients with familial hypochromic anaemia were young adults. Their age and sex appear from Table I. 7 normal individuals, 3 young adults and 4 children served as controls. Amino acid analyses in plasma and erythrocytes were performed on a Technicon Amino Acid Analyzer by the methods of Perry et a1 (1970). The blood examinations were made in the morning after an overnight fasting period of at least 10 h. EDTA was used as anticoagulant. After the buffy coat was removed, the erythrocytes were washed three times with equal volumes (3-5 ml) isotonic NaCI. The operations were carried out in a cooling room at 4 O C. The erythrocytes were haemolysed with distilled water (1:l) and the protein was precipitated with 200
180
M. SEIP, R. LINDEMANN, P. GJESDAHL & L. R. GJESSING
mg of sulfosalicylic acid. The chromatograms were calculated by the Technicon Intergratorcalculator. In two cases the glycine and the oxidized glutathione came out together which prevented their determination. Proline and hydroxyproline were not determined in the erythrocytes.
from normal except one case with Fanconi anaemia where 8 amino acids were elevated and the case with aplastic anaemia where 11 amino acids were elevated. These two cases had 7 amino acids increased in common and both had low taurine. Both were on medication with anabolic steroid and corticosteroid. Low plasma taurine was also RESULTS found in spherocytosis and one case with The results of the amino acid analyses are non-spherocytic haemolytic anaemia. tabulated in Tables 11, I11 and IV, while the Most amino acids in the erythrocytes haemoglobin, red cell and reticulocyte val- were also normal, but here there are striking ues of the patients at the time of the study exceptions, which are written with bold are given in Table I. In Tables 11, I11 and types in Tables 11, I11 and IV. IV the values for reduced and oxidized The 2 patients with high levels of plasma glutathione (GSH and GSSG) are also amino acids, one case with Fancmi and one presented. These are small peptides, not with aplastic anaemia, had 13 and 10 eryamino acids. throcyte amino acids elevated, respectively. In the controls the concentrations both The second patient with Fanconi anaemia in plasma and red blood cells, and the PA2 had 6 amino acids elevated. ratios were of the same order of magnitude Erythrocyte glutamic acid concentrations in children as in adults for almost all amino were moderately to markedly elevated in all acids. However, the plasma concentrations patients studied, both those with aregeneraof tryptophan, and the erythrocyte concen- tory and those with haemolytic anaemias. trations of aspartic acid were slightly lower The highest values, three to four times the in the children. normal average, were found in the two paIt will be seen that cystine, methionine tients with congenital sideroblastic anaemia. and tryptophan were present in plasma, but Elevated erythrocyte glycine levels were not (or in minute quantities only) in the red found in all patients except in one patient blood cells. On the other hand, aspartic with hypochromic anaemia and one with acid was mainly found in erythrocytes, very Diamond-Blackfan anaemia. Very high little in plasma, and glutathione was ex- levels were found in congenital sideroclusively present in erythrocytes. Most of blastic anaemia and in congenital spherothe other amino acids were found in higher cytosis. concentrations in plasma than in the cells, Glutamine was elevated in 8 cases: one however, glutamic acid and ornithine were with sideroblastic anaemia, both cases with more concentrated in the cells. Glycine and Fanconi anaemia, the patient with aplastic asparagine showed approximately the same anaemia, two cases with Diamond-Blackfan concentrations in erythrocytes as in plasma. anaemia, and in the patients with spheroHydroxyproline and proline were not mea- cytosis and PK-deficiency. sured in the erythrocytes. A four-fold increase of serine was found In the patients, plasma amino acid con- in one patient (sideroblastic anaemia). Othercentrations showed no or little deviation wise serine was elevated in 7 cases: both
AMINO ACID CONCENTRATIONS IN ANAEMIAS
181
TABLE I1 Free amino acids (pmoles/l) in plasma (P) and red blood cells ( E ) in normal individuals I
I
Normal adults R.L. 2 9 Age (yrs) Sex Taurine Aspartic acid Hydroxyproline Threonine Serine Asparagine Glutamic acid Glutamine Proline Glycine Alanine Citrulline wminobutyric acid Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Ornithine Lysine Histidine Arginine GSH GSSG
* = Gly + GSSG
1 K.G. 3 7
1
Normal children B.M. ;
M
L.L.S. 5 Y2 F
A.N. 7 '/2 F
P/E 173/ 83 6/281 4/ND 135/ 68 1181 72 25/ 44 12U180 601/262 157/ND 1861178 382/181 311 20
P/E 471 65 41341 14/ND 1271 64 115/ 83 5U 44 1091169 4901253 168/ND 2611226 381/217 27/ 25
P/E 201/153 9130.5 -/ND 179/ 88 1101 89 68/ 78 1571264 5971288 121/ND 251/251 637/351 22/ 16
P/E 831 54 3/236 -/ND 82/ 42 1281 18 46/ 88 39/194 48W342 NDND 1991260 218/162 461 24
PIE 42/109 1/110 33/ND 106/ 48 146/ 99 25/ 22 122/189 4611214 247/ND 274/281 259/133 24/ 11
P/E 51/101 tr/ 92 7/ND 98/ 35 144/ 84 44/ND 37/179 6901322 19UND 237/ * 262/146 28/ 3
P/E 87/218 tr/194 5/ND 115/ 55 1311 98 44/ND 15/184 5791409 140WD 220/303 282/146 19/ 6
301 18 253/ 94 53/ 27/ 651 20 132/ 28 651 37 6U 17
19/ 17 190/ 72 49/ -
22/ 16 178/ 72 531 24/ 2 40/ 15 951 26 59/ 34 521 15 44/ 53/ 89 158/ 84 98/ 52 86/ 23 -/ -/>1412
18/ 7 172/ 74 37/ 14/ 5 40/ 16 79/ 27 53/ 32 40/ 13 23/ 55/ 88 165/ 76 65/ 35 951 23 41192 4622
191 6 174/ 59 33/ 17/ 41/ 13 82/ 18 45/ 22 44/ 13 25/ 411 93 184/ 82 95/ 47 10U 13
14/ tr 190/ 35 26/ 18/ tr 531 7 86/ 10 56/ 18 45/ 4 241 41/ 21 179/ 24 761 23 1OU tr 4304 -/1104*
12/ tr 184/ 64 34/ 17/ tr 44/ 16 79/ 20 391 12 38/ 6 12/ 26/ 48 133/ 64 80/ 39 65/ 12
so/ -
44/ 49 1981 65 89/ 46 831 19 -1 -11312
22/
-
51/ 17 901 25 44/ 22 46/ 14 45/ 44/ 61 144/ 54 1031 54 71/ 18 -/ 41124
-/
-
4765
-/
-
41339
N D = not determined
the sideroblastic anaemia patients, both with Fanconi anaemia, and the patients with aplastic anaemia, spherocytosis and PKdeficiency. Asparagine was increased in red blood cells from the two patients with Fanconi anaemia, one patient with familial hypochromic anaemia, one with Diamond-Blackfan anaemia, and the patient with spherocytosis. Aspartic acid in erythrocytes was reduced in sideroblastic anaemia, and in familial hypochromic anaemia, and in one patient with haemolytic anaemia.
Threonine was elevated in one patient with sideroblastic anaemia, both with Fanconi anaemia, and in the patient with aplastic anaemia. Erythrocyte taurine concentrations were elevated in oae patient with sideroblastic anaemia, and in two with Diamond-Blackfan anaemia. Alanine was increased in one case with sideroblastic anaemia and one with Fanconi anaemia, while valine, ornithine and lysine were elevated in one patient with Fanconi anaemia and one with aplastic anaemia. Citrulline was moderately increased in
M. SEIP, R. LINDEMANN, P. GJESDAHL & L. R. GJESSING
182
TABLE 111 Free amino acids (pmoles/l) in plasma ( P ) and red blood cells ( E ) in patients with certain aregeneratory anaemias Congenital sideroblastic anaemia
Fanconi anaemia L.L.A. P/E
Taurine Aspartic acid Hy droxyproline Threonine Ssrine Asparagine Glutamic acid Glutamine Proline Glycine Alanine Citrulline a-aminobutyric acid Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Ornithine Lysine Histidine Arginine GSH GSSG
* = Gly + GSSG
I
. ; : 0
Aplastic anaemia (constitutional)
Familial hypochromic anaemia with hypersideraernia
T.J. P/E
E.H. P/E
1
E.S. P/E
55/530 8/ 80 18/ND 125/136 134/634 68/ND 661816 578/608 240/ND 1831 * 376/604 33/ 14
147/146 -/ 79 tr/ND 84/46 114/129 50Y 60 43/635 556/324 170/ND 3171867 342/178 28/ 16
57/ 31 2/163 20/ND 207/102 144/159 52/120 69/268 560/474 178/ND 250/311 553/315 11/ 13
26/ 73 21235 22/ND 193/140 1391184 46/276 39/407 573/655 27VND 247/443 446649 27/ 45
29/ 43 3/223 30/ND 356/205 205/149 42/ 48 67/359 986/682 216/ND 313/385 426/295 27/ 27
121/189 5/ 87 10/ND 130f 58 122/ 78 24/ 94 66/396 681/354 92/ND 219/480 295/194 21/ 19
151/128 8/ 47 20/ND 130/ 40 88/ 57 20/ 28 921316 668/286 23UND 233/290 402/128 28/ 4
30/ 18 269/ 98 16/ 21/ 69/ 26 105/ 40 76/ 44 49/ 22 4u 761 56 183/ 96 89/ 40 431 14 -/la28 -/1902*
22/ 8 226/ 74 19/ 13/ 38/ 8 73/ 14 43/ 20 46/ 10 31/ 741 52 1421 44 84/ 30 601 6 4264 41800
10/ 4 185/ 60 371 25/ 651 25 831 21 8CV 41 511 18 38/ 401 69 175/ 75 83/ 50 901 20 -/ 26 41347
21/ 25 244/163 54/ 30/ 851 38 174/ 55 55/ 40 53/ 21 59/ 60/124 395/155 81/ 58 1831 34 4110 -/>1640
28/ 16 2511136 66/ 341 80/ 35 183/ 57 751 40 68/ 27 59/ 89/120 395/124 107/ 49 220/ 28 -/ 42 -/>1580
33/ 30 146/ 81 45/ 19/ 37/ 21 84/ 24 39/ 31 36/ 26 33/ 33/ 66 158/ 96 113/ 41 79/ND -/ 41538
22/ 14 197/ 39 66/ 28/ 54/ 7 130/ 15 53/ 16 6UND 45/ 481 31 230/ 38 113/ 33 12lf 17 -/ 41054
N D = not determined
one patient with Fanconi anaemia and the patient with PK-deficiency. DISCUSSION
As in most earlier reports on free amino acids in erythrocytes, the values are here given per unit of packed red cells. It should be remembered that the water content of erythrocytes (about 63 %) is less than the water content of plasma (94.5 %). Expressed in relation to the water content of erythrocytes and of plasma, respectively,
the values for free amino acids in red blood cells would be approximately 50 % higher. It should also be remembered that the red cell water increases when the haemoglobin content of the cell decreases, as in hypochromic anaemias. Our amino acid values in the normal controls are in good agreement with those obtained by Levy & Barkin (1971) with a similar, though not identical method. Levy & Barkin did not measure hydroxyproline, asparagine, proline and tryptophan. Our values in red blood cells are somewhat
153
AMINO ACID CONCENTRATIONS IN ANAEMIAS TABLE IV Free amino acids (pmoles/l) in plasma ( P ) and red blood cells (E) in patients with Diarnond-Blackfan anaemia and in haemolytic anaemias
I
Diamond-Blackfan anaemia
Taurine Aspartic acid Hydroxyproline Threonine Serine Asparagine Glutamic acid Glutamine Pro1ine Glycine Alanine Citrulline a-aminobutyric acid W in e Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Ornithine Lysine Histidine Arginine GSH GSSG
Spherocytosis
1
PK deficiency
Non-spherocytic haemolytic anaemia
41/ 50 3/302 8/ND 104/ 55 138/112 42f 48 102f356 410/268 118/ND 167/318 1531112 l U 17
1601 87 6/292 8/ND 14U 73 1291114 50/ 72 129/292 612/384 169/ND 256/268 383/211 241 25
42/410 tr/ 89 13/ND 111/ 54 103/111 241 26 49/527 543/304 219/ND 204/512 445/287 15/ 8
38/250 tr/304 lOMD 154/ 66 135/ 98 31/ 38 57/359 636/433 215/ND 223/323 603/256 lU 9
2081217 12/ND 16/ND 118/ 67 99/103 16/100 73/439 690/545 124/ND 2161331 184/108 17/ 8
20/ 93 2/ 70 20/ND 147/ 98 117/183 34/184 54/275 708/478 136/ND 225/1058 303/235 25/ 22
21/ 17 242/ 93 48/ 22f 731 23 136/ 36 651 34 60/ 21 40/ 60/ 73 1781 56 10U 57 96/ 9 -/ -/>1616
25/ 15 2031 78 45/ lU 55/ 17 115/ 34 38/ 15 461 16 18/ 30/ 23 130/ 41 74/ 34 67/ 44 -/ 96 -/>1603
24/ 12 2381 80 48/ 25/ 62f 21 124/ 41 50/ 24 531 18 24/ 59/ 63 185/ 61 801 37 9U 20 4 -/>1390
30/ND 162/ 70 46/ 16/ 45/ 28 w 33 48/ 20 42/ 14 36/ 43/ 53 175/ 57 83/ 38 75/ 15 -/ND -/>990
26/ 20 11/ 17 19/ 13 224/ 78 118/ 55 124/ 47 5u 39/ 44/ 18/ 21/ 16/ 49/ 16 38/ 13 50/ 14 104/ 24 71/ 15 59/ 16 41/ 25 81/ 34 44/ 26 43/ 13 35/ 11 35/ 12 34/ 44/ 20/ 26/ 44 4WND 30/ 47 151/ 72 139/ 79 1781 90 64/ 41 77/ 31 791 48 69/ 28 78/ 27 601 19 -/ 12 -/ -/ 41522 -/>1352 -/>1612
-
46/146 4/191 18/ND 106/ 79 13W143 42/ 54 103/559 5151439 103/ND 213/461 219/229 25/ 34
I
33/115 6/228 20/ND 79/ 33 1101 81 44/ 42 103/571 3901198 117/ND 235/537 148/ 86 23/ 20 20/ 18 1791 45 431 131 461 9 84/ 18 46/ 16 441 11 301 22/ 29 136/ 48 63/ 25 68/ 11 -/
-
-/>1468
ND = not determined
lower than theirs for 9 amino acids (threonine, serine, citrulline, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine). This may possibly be due to washing of the red cells three times with normal saline in our analyses in order to remove admixture of plasma. Levy & Barkin did not wash the red cells, and a certain amount of plasma has undoubtedly been included in their analyses of red blood cells. It is not likely that the washing procedure used in our studies with small quantities of saline (3-5 ml) at 4 O C has caused significant loss of amino acids from the red
cells (Bjornesjo 1968a). McMenamy et a1 (1960a) and Nicolaidou et al (1965) using paper chromatography found on the whole somewhat higher intracellular concentrations of amino acids. The marked gradients observed across the red cell membrane for many amino acids, and the differences in P/E ratios between different amino acids, indicate that the amino acids do not enter the cells by simple diffusion, and that more selective processes are involved. One could expect that the amino acids showing the relatively highest intracellular concentrations (aspartic
184
M. SEIP, R. LINDEMANN, P. GJESDAHL & L. R. GJESSING
acid, glutamic acid, glycine, asparagine, ornithine) would be the ones most frequently showing deviations from normal in anaemias. This was also the case for glutamic acid and glycine, and to some extent aspartic acid and asparagine. In blood disorders plasma amino acid concentrations usually do not show significant deviations from normal (Mc Menamy et al (1960b), Nicolaidou et al (1965), this study). Few studies of free amino acids in red blood cells in blood disorders have been reported until now, and in all these studies paper chromatography has been used. Iyer (1958) found increases in concentrations of alanine, glutamic acid, glutamine and serine plus glycine in iron deficiency anaemia. In various types of leukaemia McMenamy et a1 (1960b) found the total unbound amino acid concentrations in erythrocytes to be approximately 10 % higher than the normal average. Most of this increase was attributable to glutamic acid and serine plus glycine. Ornithine and proline were also significantly higher in the patients. Nicolaidou et al (1965) studied 8 children with iron deficiency anaemia, 4 newborns with erythroblastosis foetalis, and 12 children with various haemolytic anaemias. They found that low haemoglobin concentrations and/ or elevated reticulocyte counts were almost always accompanied by increased concentrations of every amino acid determined in the erythrocytes. No recognizable distinction as to the disease category was evident in the unbound amino acid patterns of the anaemia patients. These latter results are in certain respects at variance with those obtained by us. In all 4 patients with haemolytic anaemias glutamic acid and glycine were moderately to markedly increased in the red blood
cells. Moderate elevations of certain other amino acids were occasionally observed (see below), but no generalized increase in erythrocyte amino acids was found. We have been unable to find any data from the literature concerning erythrocyte amino acids in aregeneratory anaemias. In this study glutamic acid was increased in all 11 patients with various types of aregeneratory anaemia, as well as in those with haemolytic anaemia. High erythrocyte glycine concentration was observed in 9 of 11 cases in this group and in all the haemolytic cases. It has been shown that glycine is easily taken up by erythrocytes, while glutamic acid is not (Bjornesjo 1965). The glycine entering the red cells largely exchanges with the glycine of the erythrocyte glutathione (Bjornesjo et a1 1968). Allen (1960) reported higher amino acid concentrations in reticvlocytes than in mature red cells, and Bjornesjo et a1 (1968) found increased uptake of glycine by erythrocytes from patients with anaemia and high reticulocyte counts. The presence of a young population of red cells might possibly explain, at least partly, the elevated levels of glutamic acid and glycine intracellularly in haemolytic anaemias, however, not in the patients with aregeneratory anaemia and normal reticulocyte counts. It seems as if the increased erythrocyte glutamic acid and glycine levels in some way are related to anaemia per se. Several hormones are known to increase the cellular uptake of amino acids (Bjornesjo 1968b). In anaemias the plasma levels of the hormone erythropoietin rise, but if this can influence the cellular uptake of amino acids is not known. Moderate elevations of erythrocyte asparagine were somewhat more common in aregeneratory (4/1 l), than in haemolytic (1/4) anaemias. The predominantly intracellular
AMINO ACID CONCENTRATIONS IN ANAEMIAS
amino acid aspartic acid, however, was decreased in 4 cases of aregeneratory and 1 case of haemolytic anaemia. Among the amino acids which normally are more concentrated in plasma than in erythrocytes, some showed elevated levels in some of the patients. These elevations were in most cases moderate. Glutamine was elevated in 6 of 11 cases of the aregeneratory and in 2 of 4 cases of the haemolytic anaemias, whereas serine was increased in 5 of the former and in 2 of the latter group. Erythrocyte taurine was high in 3/11 in the aregeneratory group. None of the observed deviations from normal erythrocyte amino acid concentrations were specific for any one type of anaemia, and determinations of erythrocyte amino acid concentrations do therefore not seem to be of differential diagnostic value. An exception might possibly be congenital sideroblastic anaemia, in which the erythrocyte glycine and glutamic acid concentrations were particularly high. The elevations found for many amino acids both in plasma and erythrocytes in 1 patient with Fanconi anaemia and 1 with (constitutional?) aplastic anaemia may possibly be related, at least partly, to the treatment, since they both received an anabolic steroid plus prednisone. It should be mentioned, however, that the second patient with Fanconi anaemia, who was not on hormone therapy, also had 6 amino acids elevated intracellularly. Further studies on unbound amino acids in erythrocytes and the uptake of amino acids by erythrocytes under normal and pathologic conditions may be of value thereby that they could provide important information concerning red blood cell metabolism.
185
ACKNOWLEDGEMENT We are grateful to Mr. Reidar Langseth for excellent technical assistance.
REFERENCES Allen D W (1960) Amino acid accumulation by human reticulocytes. Blood 16, 1564-71. Barber G W & Spaeth G L (1969) The successful treatment of homocystinuria with pyridoxine. J Pediat 75, 463-78. Bjornesjo K B (1965) Uptake of labelled amino acids into human erythrocytes in vitro. Clin Chim Actu 11, 197-204. Bjornesjo K B (1968a) The distribution of amino acids between erythrocytes and plasma in fetal and maternal blood. Clin Chim Acta 20, 11-15. Bjomesjo K B (1968b) The erythrocytdplasma distribution of amino acids in health and disease. Clin Chim Actu 20, 17-22. Bjornesjo K B, Jarnulf B & Lausing E (1968) Uptake of labelled amino acids into human erythrocytes in disease. Clin Chim Actu 20, 2327. Iyer G Y W (1958) Free amino acid levels in normal and anemic erythrocytes in relation to their potassium content and rate of glycolysis. Arch Biochem Biophys 74, 24-27. Levy H L & Barkin L (1971) Comparison of amino acid concentrations between plasma and erythrocytes. Studies in normal subjects and those with metabolic disorders. J Lab Clin Med 87, 517-23. McMenamy R H, Lund C C, Neville G J & Wallach D F H (1960a) Studies of unbound amino acid distributions in plasma, erythrocytes, leukocytes and urine of normal human subjects. J Clin Invest 39, 1675-87. McMenamy R H, Lund C C & Wallach D F H (1960b) Unbound amino acid concentrations in plasma, erythrocytes, leukocytes and urine of patients with leukemia. J Clin Znvest 39, 1688-1705. Nicolaidou M, Lund C C & McMenamy R H (1965) Unbound amino acids in the plasma and erythrocytes of children with various anemias and with erythroblastosis fetalis. Blood 26, 9199. Perry T L, Hansen S, Tischler B, Bunting R &
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Diamond S (1970) Glutamine depletion in phenylketonuria. N Engl J Med 282, 761-66. Seip M, Gjessing L R & Lie S 0 (1971) Congenital sideroblastic anaemia in a girl. S c a d J Haematol 8, 505-12.
Soupart P (1962) Free amino acids of blood and urine in the human. In J T Holden (ed) Amino Acid Pools, pp 220-62. Elsevier Publishing Company, Amsterdam.
Amino Acid Concentrations in Plasma and Erythrocytes in Aregeneratory and Haemolytic Anaemias MARTIN SEIP,M.D., ROLFLINDEMANN, M.D., PERGJESDAHL, Civ. Eng., & LEIVR.GJESSING, M.D. Pediatric Research Institute and Department of Pediatrics (Chief, M . Seip), Rikshospitalet, Oslo, and Dikernark Hospital, Asker, Norway
The concentrations of unbound amino acids in erythrocytes and in plasma from 7 normal individuals, 11 patients with various types of aregeneratory anaemia, and 4 patients with hereditary haemolytic anaemias were determined on a Technicon Amino Acid Analyzer (Perry et a1 1970). Most amino acids were normally found in higher concentrations in plasma than intracellularly. Cystine, methionine and tryptophan were almost exclusively present in plasma. Aspartic acid, however, was mainly found in erythrocytes, and glutathione only in erythrocytes. Glutamic acid and ornithine were more concentrated in the cells, while glycine and asparagine showed approximately the same concentrations in erythrocytes as in plasma. In the patients, plasma amino acids showed little deviations from normal, but in the erythrocytes there were striking changes. Erythrocyte glutamic acid concentrations were moderately to markedly elevated in all patients studied, and glycine concentrations in 13 out of 15 patients. In addition, the following amino acids were increased intracellularly in more than one patient: glutamine (8 patients), serine (7), asparagine (3,threonine (4), taurine (3), alanine (2), valine (2), ornithine (2), lysine (2), citrulline (2). Aspartic acid was decreased in erythrocytes from 4 patients with aregeneratory and 1 with haemolytic anaemia. Key words: amino acids - plasma
- erythrocytes - aregenerative anaemia
Accepted for publication June 24, 1975 Correspondence to: Prof. Martin Seip, Department of Pediatrics, Rikshospitalet, Oslo 1, Norway
Although much information is available concerning plasma concentrations of amino acids in health and disease, our knowledge of free amino acid content of red blood cells is sparse. A few authors have reported quantitative studies of amino acids in erythrocytes in normal individuals (McMenamy
et al 1960a/b, Soupart 1962, Levy & Barkin 1971), and in patients with various metabolic disorders (Barber & Spaeth 1969, Levy & Barkin 1971). Semiquantitative determinations of amino acids in red blood cells of healthy and diseased adults, and of foetal and maternal blood have also been
AMINO ACID CONCENTRATIONS IN ANAEMIAS
reported (Bj omesjo 1968ah). A few investigators have studied plasma and erythrocyte concentrations of unbound amino acids in various blood disorders, leukaemia (McMenamy et a1 1960b), haemolytic anaemias (Allen 1960), iron deficiency anaemia and haemolytic anaemias (Nicolaidou et a1 1965). Our interest in these problems was aroused during the study of two girls with congenital sideroblastic anaemia probably due to deltaaminolaevulinic acid (ALA) synthetase deficiency (Seip et a1 1971). Both patients showed very high levels of glycine and glutamic acid in their erythrocytes. The intention of this work was to study free amino acid Concentrations in erythrocytes of patients with aregeneratory and haemolytic anaemia, and to compare with the plasma concentrations. -03
;Fr,5\dA I
MATERIAL AND METHODS The patient material (Table I) consists of 11 patients with aregeneratory anaemia. The 4 patients with Diamond-Blackfan anaemia were on prednisone medication, while 1 patient with Fanconi anaemia and 1 with aplastic anaemia received anabolic steroids plus prednisone. In addition 4 patients with congenital haemolytic anaemia
were included. Most of the patients were children, however, 2 patients with familial hypochromic anaemia were young adults. Their age and sex appear from Table I. 7 normal individuals, 3 young adults and 4 children served as controls. Amino acid analyses in plasma and erythrocytes were performed on a Technicon Amino Acid Analyzer by the methods of Perry et a1 (1970). The blood examinations were made in the morning after an overnight fasting period of at least 10 h. EDTA was used as anticoagulant. After the buffy coat was removed, the erythrocytes were washed three times with equal volumes (3-5 ml) isotonic NaCI. The operations were carried out in a cooling room at 4 O C. The erythrocytes were haemolysed with distilled water (1:l) and the protein was precipitated with 200
180
M. SEIP, R. LINDEMANN, P. GJESDAHL & L. R. GJESSING
mg of sulfosalicylic acid. The chromatograms were calculated by the Technicon Intergratorcalculator. In two cases the glycine and the oxidized glutathione came out together which prevented their determination. Proline and hydroxyproline were not determined in the erythrocytes.
from normal except one case with Fanconi anaemia where 8 amino acids were elevated and the case with aplastic anaemia where 11 amino acids were elevated. These two cases had 7 amino acids increased in common and both had low taurine. Both were on medication with anabolic steroid and corticosteroid. Low plasma taurine was also RESULTS found in spherocytosis and one case with The results of the amino acid analyses are non-spherocytic haemolytic anaemia. tabulated in Tables 11, I11 and IV, while the Most amino acids in the erythrocytes haemoglobin, red cell and reticulocyte val- were also normal, but here there are striking ues of the patients at the time of the study exceptions, which are written with bold are given in Table I. In Tables 11, I11 and types in Tables 11, I11 and IV. IV the values for reduced and oxidized The 2 patients with high levels of plasma glutathione (GSH and GSSG) are also amino acids, one case with Fancmi and one presented. These are small peptides, not with aplastic anaemia, had 13 and 10 eryamino acids. throcyte amino acids elevated, respectively. In the controls the concentrations both The second patient with Fanconi anaemia in plasma and red blood cells, and the PA2 had 6 amino acids elevated. ratios were of the same order of magnitude Erythrocyte glutamic acid concentrations in children as in adults for almost all amino were moderately to markedly elevated in all acids. However, the plasma concentrations patients studied, both those with aregeneraof tryptophan, and the erythrocyte concen- tory and those with haemolytic anaemias. trations of aspartic acid were slightly lower The highest values, three to four times the in the children. normal average, were found in the two paIt will be seen that cystine, methionine tients with congenital sideroblastic anaemia. and tryptophan were present in plasma, but Elevated erythrocyte glycine levels were not (or in minute quantities only) in the red found in all patients except in one patient blood cells. On the other hand, aspartic with hypochromic anaemia and one with acid was mainly found in erythrocytes, very Diamond-Blackfan anaemia. Very high little in plasma, and glutathione was ex- levels were found in congenital sideroclusively present in erythrocytes. Most of blastic anaemia and in congenital spherothe other amino acids were found in higher cytosis. concentrations in plasma than in the cells, Glutamine was elevated in 8 cases: one however, glutamic acid and ornithine were with sideroblastic anaemia, both cases with more concentrated in the cells. Glycine and Fanconi anaemia, the patient with aplastic asparagine showed approximately the same anaemia, two cases with Diamond-Blackfan concentrations in erythrocytes as in plasma. anaemia, and in the patients with spheroHydroxyproline and proline were not mea- cytosis and PK-deficiency. sured in the erythrocytes. A four-fold increase of serine was found In the patients, plasma amino acid con- in one patient (sideroblastic anaemia). Othercentrations showed no or little deviation wise serine was elevated in 7 cases: both
AMINO ACID CONCENTRATIONS IN ANAEMIAS
181
TABLE I1 Free amino acids (pmoles/l) in plasma (P) and red blood cells ( E ) in normal individuals I
I
Normal adults R.L. 2 9 Age (yrs) Sex Taurine Aspartic acid Hydroxyproline Threonine Serine Asparagine Glutamic acid Glutamine Proline Glycine Alanine Citrulline wminobutyric acid Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Ornithine Lysine Histidine Arginine GSH GSSG
* = Gly + GSSG
1 K.G. 3 7
1
Normal children B.M. ;
M
L.L.S. 5 Y2 F
A.N. 7 '/2 F
P/E 173/ 83 6/281 4/ND 135/ 68 1181 72 25/ 44 12U180 601/262 157/ND 1861178 382/181 311 20
P/E 471 65 41341 14/ND 1271 64 115/ 83 5U 44 1091169 4901253 168/ND 2611226 381/217 27/ 25
P/E 201/153 9130.5 -/ND 179/ 88 1101 89 68/ 78 1571264 5971288 121/ND 251/251 637/351 22/ 16
P/E 831 54 3/236 -/ND 82/ 42 1281 18 46/ 88 39/194 48W342 NDND 1991260 218/162 461 24
PIE 42/109 1/110 33/ND 106/ 48 146/ 99 25/ 22 122/189 4611214 247/ND 274/281 259/133 24/ 11
P/E 51/101 tr/ 92 7/ND 98/ 35 144/ 84 44/ND 37/179 6901322 19UND 237/ * 262/146 28/ 3
P/E 87/218 tr/194 5/ND 115/ 55 1311 98 44/ND 15/184 5791409 140WD 220/303 282/146 19/ 6
301 18 253/ 94 53/ 27/ 651 20 132/ 28 651 37 6U 17
19/ 17 190/ 72 49/ -
22/ 16 178/ 72 531 24/ 2 40/ 15 951 26 59/ 34 521 15 44/ 53/ 89 158/ 84 98/ 52 86/ 23 -/ -/>1412
18/ 7 172/ 74 37/ 14/ 5 40/ 16 79/ 27 53/ 32 40/ 13 23/ 55/ 88 165/ 76 65/ 35 951 23 41192 4622
191 6 174/ 59 33/ 17/ 41/ 13 82/ 18 45/ 22 44/ 13 25/ 411 93 184/ 82 95/ 47 10U 13
14/ tr 190/ 35 26/ 18/ tr 531 7 86/ 10 56/ 18 45/ 4 241 41/ 21 179/ 24 761 23 1OU tr 4304 -/1104*
12/ tr 184/ 64 34/ 17/ tr 44/ 16 79/ 20 391 12 38/ 6 12/ 26/ 48 133/ 64 80/ 39 65/ 12
so/ -
44/ 49 1981 65 89/ 46 831 19 -1 -11312
22/
-
51/ 17 901 25 44/ 22 46/ 14 45/ 44/ 61 144/ 54 1031 54 71/ 18 -/ 41124
-/
-
4765
-/
-
41339
N D = not determined
the sideroblastic anaemia patients, both with Fanconi anaemia, and the patients with aplastic anaemia, spherocytosis and PKdeficiency. Asparagine was increased in red blood cells from the two patients with Fanconi anaemia, one patient with familial hypochromic anaemia, one with Diamond-Blackfan anaemia, and the patient with spherocytosis. Aspartic acid in erythrocytes was reduced in sideroblastic anaemia, and in familial hypochromic anaemia, and in one patient with haemolytic anaemia.
Threonine was elevated in one patient with sideroblastic anaemia, both with Fanconi anaemia, and in the patient with aplastic anaemia. Erythrocyte taurine concentrations were elevated in oae patient with sideroblastic anaemia, and in two with Diamond-Blackfan anaemia. Alanine was increased in one case with sideroblastic anaemia and one with Fanconi anaemia, while valine, ornithine and lysine were elevated in one patient with Fanconi anaemia and one with aplastic anaemia. Citrulline was moderately increased in
M. SEIP, R. LINDEMANN, P. GJESDAHL & L. R. GJESSING
182
TABLE 111 Free amino acids (pmoles/l) in plasma ( P ) and red blood cells ( E ) in patients with certain aregeneratory anaemias Congenital sideroblastic anaemia
Fanconi anaemia L.L.A. P/E
Taurine Aspartic acid Hy droxyproline Threonine Ssrine Asparagine Glutamic acid Glutamine Proline Glycine Alanine Citrulline a-aminobutyric acid Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Ornithine Lysine Histidine Arginine GSH GSSG
* = Gly + GSSG
I
. ; : 0
Aplastic anaemia (constitutional)
Familial hypochromic anaemia with hypersideraernia
T.J. P/E
E.H. P/E
1
E.S. P/E
55/530 8/ 80 18/ND 125/136 134/634 68/ND 661816 578/608 240/ND 1831 * 376/604 33/ 14
147/146 -/ 79 tr/ND 84/46 114/129 50Y 60 43/635 556/324 170/ND 3171867 342/178 28/ 16
57/ 31 2/163 20/ND 207/102 144/159 52/120 69/268 560/474 178/ND 250/311 553/315 11/ 13
26/ 73 21235 22/ND 193/140 1391184 46/276 39/407 573/655 27VND 247/443 446649 27/ 45
29/ 43 3/223 30/ND 356/205 205/149 42/ 48 67/359 986/682 216/ND 313/385 426/295 27/ 27
121/189 5/ 87 10/ND 130f 58 122/ 78 24/ 94 66/396 681/354 92/ND 219/480 295/194 21/ 19
151/128 8/ 47 20/ND 130/ 40 88/ 57 20/ 28 921316 668/286 23UND 233/290 402/128 28/ 4
30/ 18 269/ 98 16/ 21/ 69/ 26 105/ 40 76/ 44 49/ 22 4u 761 56 183/ 96 89/ 40 431 14 -/la28 -/1902*
22/ 8 226/ 74 19/ 13/ 38/ 8 73/ 14 43/ 20 46/ 10 31/ 741 52 1421 44 84/ 30 601 6 4264 41800
10/ 4 185/ 60 371 25/ 651 25 831 21 8CV 41 511 18 38/ 401 69 175/ 75 83/ 50 901 20 -/ 26 41347
21/ 25 244/163 54/ 30/ 851 38 174/ 55 55/ 40 53/ 21 59/ 60/124 395/155 81/ 58 1831 34 4110 -/>1640
28/ 16 2511136 66/ 341 80/ 35 183/ 57 751 40 68/ 27 59/ 89/120 395/124 107/ 49 220/ 28 -/ 42 -/>1580
33/ 30 146/ 81 45/ 19/ 37/ 21 84/ 24 39/ 31 36/ 26 33/ 33/ 66 158/ 96 113/ 41 79/ND -/ 41538
22/ 14 197/ 39 66/ 28/ 54/ 7 130/ 15 53/ 16 6UND 45/ 481 31 230/ 38 113/ 33 12lf 17 -/ 41054
N D = not determined
one patient with Fanconi anaemia and the patient with PK-deficiency. DISCUSSION
As in most earlier reports on free amino acids in erythrocytes, the values are here given per unit of packed red cells. It should be remembered that the water content of erythrocytes (about 63 %) is less than the water content of plasma (94.5 %). Expressed in relation to the water content of erythrocytes and of plasma, respectively,
the values for free amino acids in red blood cells would be approximately 50 % higher. It should also be remembered that the red cell water increases when the haemoglobin content of the cell decreases, as in hypochromic anaemias. Our amino acid values in the normal controls are in good agreement with those obtained by Levy & Barkin (1971) with a similar, though not identical method. Levy & Barkin did not measure hydroxyproline, asparagine, proline and tryptophan. Our values in red blood cells are somewhat
153
AMINO ACID CONCENTRATIONS IN ANAEMIAS TABLE IV Free amino acids (pmoles/l) in plasma ( P ) and red blood cells (E) in patients with Diarnond-Blackfan anaemia and in haemolytic anaemias
I
Diamond-Blackfan anaemia
Taurine Aspartic acid Hydroxyproline Threonine Serine Asparagine Glutamic acid Glutamine Pro1ine Glycine Alanine Citrulline a-aminobutyric acid W in e Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Ornithine Lysine Histidine Arginine GSH GSSG
Spherocytosis
1
PK deficiency
Non-spherocytic haemolytic anaemia
41/ 50 3/302 8/ND 104/ 55 138/112 42f 48 102f356 410/268 118/ND 167/318 1531112 l U 17
1601 87 6/292 8/ND 14U 73 1291114 50/ 72 129/292 612/384 169/ND 256/268 383/211 241 25
42/410 tr/ 89 13/ND 111/ 54 103/111 241 26 49/527 543/304 219/ND 204/512 445/287 15/ 8
38/250 tr/304 lOMD 154/ 66 135/ 98 31/ 38 57/359 636/433 215/ND 223/323 603/256 lU 9
2081217 12/ND 16/ND 118/ 67 99/103 16/100 73/439 690/545 124/ND 2161331 184/108 17/ 8
20/ 93 2/ 70 20/ND 147/ 98 117/183 34/184 54/275 708/478 136/ND 225/1058 303/235 25/ 22
21/ 17 242/ 93 48/ 22f 731 23 136/ 36 651 34 60/ 21 40/ 60/ 73 1781 56 10U 57 96/ 9 -/ -/>1616
25/ 15 2031 78 45/ lU 55/ 17 115/ 34 38/ 15 461 16 18/ 30/ 23 130/ 41 74/ 34 67/ 44 -/ 96 -/>1603
24/ 12 2381 80 48/ 25/ 62f 21 124/ 41 50/ 24 531 18 24/ 59/ 63 185/ 61 801 37 9U 20 4 -/>1390
30/ND 162/ 70 46/ 16/ 45/ 28 w 33 48/ 20 42/ 14 36/ 43/ 53 175/ 57 83/ 38 75/ 15 -/ND -/>990
26/ 20 11/ 17 19/ 13 224/ 78 118/ 55 124/ 47 5u 39/ 44/ 18/ 21/ 16/ 49/ 16 38/ 13 50/ 14 104/ 24 71/ 15 59/ 16 41/ 25 81/ 34 44/ 26 43/ 13 35/ 11 35/ 12 34/ 44/ 20/ 26/ 44 4WND 30/ 47 151/ 72 139/ 79 1781 90 64/ 41 77/ 31 791 48 69/ 28 78/ 27 601 19 -/ 12 -/ -/ 41522 -/>1352 -/>1612
-
46/146 4/191 18/ND 106/ 79 13W143 42/ 54 103/559 5151439 103/ND 213/461 219/229 25/ 34
I
33/115 6/228 20/ND 79/ 33 1101 81 44/ 42 103/571 3901198 117/ND 235/537 148/ 86 23/ 20 20/ 18 1791 45 431 131 461 9 84/ 18 46/ 16 441 11 301 22/ 29 136/ 48 63/ 25 68/ 11 -/
-
-/>1468
ND = not determined
lower than theirs for 9 amino acids (threonine, serine, citrulline, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine). This may possibly be due to washing of the red cells three times with normal saline in our analyses in order to remove admixture of plasma. Levy & Barkin did not wash the red cells, and a certain amount of plasma has undoubtedly been included in their analyses of red blood cells. It is not likely that the washing procedure used in our studies with small quantities of saline (3-5 ml) at 4 O C has caused significant loss of amino acids from the red
cells (Bjornesjo 1968a). McMenamy et a1 (1960a) and Nicolaidou et al (1965) using paper chromatography found on the whole somewhat higher intracellular concentrations of amino acids. The marked gradients observed across the red cell membrane for many amino acids, and the differences in P/E ratios between different amino acids, indicate that the amino acids do not enter the cells by simple diffusion, and that more selective processes are involved. One could expect that the amino acids showing the relatively highest intracellular concentrations (aspartic
184
M. SEIP, R. LINDEMANN, P. GJESDAHL & L. R. GJESSING
acid, glutamic acid, glycine, asparagine, ornithine) would be the ones most frequently showing deviations from normal in anaemias. This was also the case for glutamic acid and glycine, and to some extent aspartic acid and asparagine. In blood disorders plasma amino acid concentrations usually do not show significant deviations from normal (Mc Menamy et al (1960b), Nicolaidou et al (1965), this study). Few studies of free amino acids in red blood cells in blood disorders have been reported until now, and in all these studies paper chromatography has been used. Iyer (1958) found increases in concentrations of alanine, glutamic acid, glutamine and serine plus glycine in iron deficiency anaemia. In various types of leukaemia McMenamy et a1 (1960b) found the total unbound amino acid concentrations in erythrocytes to be approximately 10 % higher than the normal average. Most of this increase was attributable to glutamic acid and serine plus glycine. Ornithine and proline were also significantly higher in the patients. Nicolaidou et al (1965) studied 8 children with iron deficiency anaemia, 4 newborns with erythroblastosis foetalis, and 12 children with various haemolytic anaemias. They found that low haemoglobin concentrations and/ or elevated reticulocyte counts were almost always accompanied by increased concentrations of every amino acid determined in the erythrocytes. No recognizable distinction as to the disease category was evident in the unbound amino acid patterns of the anaemia patients. These latter results are in certain respects at variance with those obtained by us. In all 4 patients with haemolytic anaemias glutamic acid and glycine were moderately to markedly increased in the red blood
cells. Moderate elevations of certain other amino acids were occasionally observed (see below), but no generalized increase in erythrocyte amino acids was found. We have been unable to find any data from the literature concerning erythrocyte amino acids in aregeneratory anaemias. In this study glutamic acid was increased in all 11 patients with various types of aregeneratory anaemia, as well as in those with haemolytic anaemia. High erythrocyte glycine concentration was observed in 9 of 11 cases in this group and in all the haemolytic cases. It has been shown that glycine is easily taken up by erythrocytes, while glutamic acid is not (Bjornesjo 1965). The glycine entering the red cells largely exchanges with the glycine of the erythrocyte glutathione (Bjornesjo et a1 1968). Allen (1960) reported higher amino acid concentrations in reticvlocytes than in mature red cells, and Bjornesjo et a1 (1968) found increased uptake of glycine by erythrocytes from patients with anaemia and high reticulocyte counts. The presence of a young population of red cells might possibly explain, at least partly, the elevated levels of glutamic acid and glycine intracellularly in haemolytic anaemias, however, not in the patients with aregeneratory anaemia and normal reticulocyte counts. It seems as if the increased erythrocyte glutamic acid and glycine levels in some way are related to anaemia per se. Several hormones are known to increase the cellular uptake of amino acids (Bjornesjo 1968b). In anaemias the plasma levels of the hormone erythropoietin rise, but if this can influence the cellular uptake of amino acids is not known. Moderate elevations of erythrocyte asparagine were somewhat more common in aregeneratory (4/1 l), than in haemolytic (1/4) anaemias. The predominantly intracellular
AMINO ACID CONCENTRATIONS IN ANAEMIAS
amino acid aspartic acid, however, was decreased in 4 cases of aregeneratory and 1 case of haemolytic anaemia. Among the amino acids which normally are more concentrated in plasma than in erythrocytes, some showed elevated levels in some of the patients. These elevations were in most cases moderate. Glutamine was elevated in 6 of 11 cases of the aregeneratory and in 2 of 4 cases of the haemolytic anaemias, whereas serine was increased in 5 of the former and in 2 of the latter group. Erythrocyte taurine was high in 3/11 in the aregeneratory group. None of the observed deviations from normal erythrocyte amino acid concentrations were specific for any one type of anaemia, and determinations of erythrocyte amino acid concentrations do therefore not seem to be of differential diagnostic value. An exception might possibly be congenital sideroblastic anaemia, in which the erythrocyte glycine and glutamic acid concentrations were particularly high. The elevations found for many amino acids both in plasma and erythrocytes in 1 patient with Fanconi anaemia and 1 with (constitutional?) aplastic anaemia may possibly be related, at least partly, to the treatment, since they both received an anabolic steroid plus prednisone. It should be mentioned, however, that the second patient with Fanconi anaemia, who was not on hormone therapy, also had 6 amino acids elevated intracellularly. Further studies on unbound amino acids in erythrocytes and the uptake of amino acids by erythrocytes under normal and pathologic conditions may be of value thereby that they could provide important information concerning red blood cell metabolism.
185
ACKNOWLEDGEMENT We are grateful to Mr. Reidar Langseth for excellent technical assistance.
REFERENCES Allen D W (1960) Amino acid accumulation by human reticulocytes. Blood 16, 1564-71. Barber G W & Spaeth G L (1969) The successful treatment of homocystinuria with pyridoxine. J Pediat 75, 463-78. Bjornesjo K B (1965) Uptake of labelled amino acids into human erythrocytes in vitro. Clin Chim Actu 11, 197-204. Bjornesjo K B (1968a) The distribution of amino acids between erythrocytes and plasma in fetal and maternal blood. Clin Chim Acta 20, 11-15. Bjomesjo K B (1968b) The erythrocytdplasma distribution of amino acids in health and disease. Clin Chim Actu 20, 17-22. Bjornesjo K B, Jarnulf B & Lausing E (1968) Uptake of labelled amino acids into human erythrocytes in disease. Clin Chim Actu 20, 2327. Iyer G Y W (1958) Free amino acid levels in normal and anemic erythrocytes in relation to their potassium content and rate of glycolysis. Arch Biochem Biophys 74, 24-27. Levy H L & Barkin L (1971) Comparison of amino acid concentrations between plasma and erythrocytes. Studies in normal subjects and those with metabolic disorders. J Lab Clin Med 87, 517-23. McMenamy R H, Lund C C, Neville G J & Wallach D F H (1960a) Studies of unbound amino acid distributions in plasma, erythrocytes, leukocytes and urine of normal human subjects. J Clin Invest 39, 1675-87. McMenamy R H, Lund C C & Wallach D F H (1960b) Unbound amino acid concentrations in plasma, erythrocytes, leukocytes and urine of patients with leukemia. J Clin Znvest 39, 1688-1705. Nicolaidou M, Lund C C & McMenamy R H (1965) Unbound amino acids in the plasma and erythrocytes of children with various anemias and with erythroblastosis fetalis. Blood 26, 9199. Perry T L, Hansen S, Tischler B, Bunting R &
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Diamond S (1970) Glutamine depletion in phenylketonuria. N Engl J Med 282, 761-66. Seip M, Gjessing L R & Lie S 0 (1971) Congenital sideroblastic anaemia in a girl. S c a d J Haematol 8, 505-12.
Soupart P (1962) Free amino acids of blood and urine in the human. In J T Holden (ed) Amino Acid Pools, pp 220-62. Elsevier Publishing Company, Amsterdam.
