Acta Pzdiatr Scand 67: 659-663, 1978
PLASMA FREE AMINO ACID CONCENTRATIONS OF BREAST-FED INFANTS B. S. LINDBLAD, G . ALFVEN and R. ZETTERSTROM From the Department of Paediatrics, Karolinska Institutet, St Goran’s Children’s Hospital, Stockholm, Sweden
ABSTRACT. Lindblad, B. S., Alfven, G . and Zetterstrom, R. (Department of Paediatrics of Karolinska Institutet at St Goran’s Children’s Hospital, Stockholm, Sweden). Plasma free amino acid concentrations of breast-fed infants. Acta Paediatr Scand, 67: 659, 1978.Photometric determination of alpha-amino nitrogen in peripheral venous plasma and urine from 20 healthy, full-term infants, 1-5 months of age, showing normal growth and development during an uncomplicated lactation, revealed lower plasma levels than what has been found in adults, or 3.7k1.1 mg/100 ml, and a urinary excretion of 41k14 mg/24 hours. Ion-exchange chromatography of deproteinized peripheral venous plasma showed low valine concentrations, an increased glycine/valine ratio and high cystine and very high taurine levels when compared to the levels of healthy American infants of comparable ages fed 3-3.5 gtkg of cow-milk protein. The findings indicate that a formula based on cow-milk protein should optimally contain only 1.0-1.2 g protein/100 ml provided that it is “humanized” not only with regard to the lactalbumin/casein ratio, but also to the cystine and taurine content. The pattern of the plasma concentrations of free amino acids reported in the present investigation may be used as a normal reference for breast-fed infants. KEY WORDS: Amino acids, plasma, breast-feeding, human milk, cow-milk formula, valine, cystine, taurine, protein requirement
The free amino acid concentrations of plasma, as measured by ion-exchange chromatography, show little inter-individual variation if certain precautions are met with in the sampling, storage and analytical procedures (30). However, the plasma levels are dependent on age and sex (2) and on the protein intake (1, 16, 31). There are four investigations known to the authors where the plasma levels of free amino acids during childhood have been correlated to age and to dietary intake; two in premature infants (27, 28), one in full-term infants during formula feeding (31) and one in children 7-13 years of age (17). In view of the increasing need for amino acid analysis of plasma during screening, diagnosis and follow-up of inborn errors of amino acid metabolism (19), during the monitoring of total parenteral nutrition (22) and during the early diagnosis of nutritional inadequacy (23), there is obviously a need to establish the levels of breast-fed full-term infants, as opposed to
those of infants fed cow-milk formula with a considerably higher protein intake. The present investigation therefore aims at determining the post-alimentary free amino acid levels of deproteinized peripheral blood plasma in normal human infants, as represented by healthy full-term infants showing normal growth and development during an uncomplicated lactation. MATERIAL AND METHODS Twenty mothers, of a mean age of 24 years, who were selling their excess breast-milk to the Stockholm milk distribution centre were hospitalized with their healthy 1 to 5-month-old (mean 80-day old) infants. The mothers’ total milk volumes were 1244k381 m1/24 hours, according to test-weighing and mechanical pumping of excess milk at home before admission. The infants, 10 girls and 10 boys, were all full-term and showed a normal growth from normal birth weights and heights and were healthy with a normal development. They had all been fed ad libitum from the breast. Supported by a grant from the Swedish Medical Research Council (no. 2583). Aclu Paediutr Scand 67
660
B . S . Lindblad et al.
UMOL/L
PnOLlL
J5U
350
500
300
250
250
200
200
150
150
100
100
50
50
PRO RUI
LYS
SER
GLY
G U
LEU
CYS
THR
VAL
ORN
TAU
ASN
PIR
ARC
HIS
GLU
ILEU
ClTR
ME
Fig. 1. The free amino acid levels of peripheral venous blood plasma of 1 to S-monthold, breast-fed human infants. Mean ? S.D. are given.
ASP
MET
5
Blood was collected from the cubital vein in a heparinized tube before the early morning feed. The blood sample was centrifuged immediately; the plasma was deproteinized with crystalline sulphosalicylic acid, 50 mg/ ml, and the supernatant stored in -72°C until analyzed for alpha-amino nitrogen (29), and for individual free amino acids on a Bio-Cal 200 (Munchen) automatic amino acid analyser according to the method of Spackman et al. (32). A lithium buffer system (3) was used, allowing for the separation of serine, glutamine and asparagine. Tryptophan is lost during deproteinization and analysis, a s it is partly bound to albumin, and is not determined by this method. Cysteine was oxidized to cystine by allowing the sample to stand in room temperature at pH 7 for 4 hours. The methionine-sulphone peak was added to the one representing methionine during the estimation of the methionine concentration. Reproducibility was mean 4.9k0.5 (SE)% for the 23 parameters determined by ionexchange chromatography, ranging from 1.2 % (glutamine) to 9.4 7% (cystine). Urine was collected during a 24-hour period. The u r h e was kept in a refrigerator during collection and stored in -72°C until analyzed for alpha-amino nitrogen content (29). I
RESULTS /
The alpha-amino nitrogen concentration of plasma during uncomplicated lactation was foundtobe3.7k1.1 ( n = l l ) rng/lOOrnlandthe urinary excretion 41? 14 ( n=20) mg/24 hours. The individual free amino acid levels and the urea concentrations are given in Table 1. The results are also given in the form of an “arninogram” in Fig. 1 , which can serve as a Actu Puediutr Scund 67
reference chart on which to plot the results of plasma amino acid analyses during infancy. DISCUSSION The alpha-amino nitrogen concentration of plasma found in the present investigation is Table 1 . The plasma free amino acid and urea concentrations of breast-fed infants 1-5 months of age ~
~
Amino acid Taurine Aspartic acid Threonine Serine Glutamine Asparagine Glutamic acid Citrulline Proline Glycine Alanine 1/2 Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine Urea
pmol/l
S.D.
n
S.E.
90 15 108 1.56 663 43 63 23 195 187 269 98 113 21 4.5 89 49 31 102 119 84 83
34 7 50 56 208 18 27 13 90 66 96 39 47 8 16 23 25 15 38 61 22 29
19 19 19 19 19 18 19 6 19 19 19 19 18 18 19 19 19 17 16 19 19 16
8 2 11 13 48 4 6 5 21 15 22 9 I1 2 4 5 6 4 10 14 5 7
2 984
1613
19
375
Plasma amino acids in infants Table 2. Plasma valine concentrations during childhood Results which can be regarded as normal plasma levels for age are in italics
Investigation (reference) Newborn infants before feeding (24) Premature babies 1-2 weeks old (28) Human milk Formula 1.8 gll00 ml Formula 2.3 gll00 ml Premature babies less than 8 weeks old (27) 170 ml human milk/kg (800 pmol VAL/kg) Formula 1.5 g/lOO ml(l300 pmol VAL/kg) Formula 3 g/ I00 ml(2 500 pmol VAL/kg) 1-5 months ofage, human milk (present investigation) Ninth day of life, formula feeding (16) 1-3 months of age, formula feeding (3 1) 1.3 g proteinlkg 1.5 g proteinlkg 1.7 g protein/kg 3-3.5 g protein/kg 9 g protein/kg 4 days - 1 year of age (25) 4 days - 12 years of age (25) 4 months-2.5 years of age (16) 1-5 years of age, Kwashiorkor (18) 7-13 years of age (17) 0.21 g proteinikg 1.9g protein/kg
3.1 g protein/kg 6-18 years of age (2) Adults (2)
Result pmol/l, M f 2 S.E.
122f 10 139k34 219f36 274f44
120 180
280
113f22 247+38
91 111
167 194k 18 592 153f 6 171f 4 222k21 66f 6
194f55 212 +50 457+32 223f 7 252f 8
slightly lower than that reported for adults (4). Moreover, the individual amino acid levels of plasma SHOW considerably lower branchchained (valine, leucine, isoleucine) amino acid levels in comparison with those of older children and adults (2, 5 , 17, 25). The dependency on age of the valine plasma levels on the one hand, and on dietary protein intake on the other, is quite evident from the review of the literature summarized in Table 2. When the present results are compared with the plasma levels of free amino acids of “control” American infants of 1-3 months of age
661
being fed cow-milk based formula in amounts 3-3.5 g protein/kg in an investigation demonstrating the effect of overfeeding (9 g protein/ kg) on the plasma “aminogram” (31) it is clear that even the “controls” show the high valine levels and decreased glycinelvaline quotient characteristic of protein overnutrition (23, 3 1). This difference between normal breastfed Swedish young infants consuming 0.82 g protein/100 ml (21) and normal cow-milk formulafed American young infants consuming 2-2.3 g protein/100 ml (Fig. 2) could be of importance, as there are indications that the branchchained amino acid and tyrosine levels of plasma in particular influence insulin metabolism. The branch-chained amino acid levels of plasma seem to be especially sensitive to exogenous and endogenous insulin (6, 9, 10, 26, 35). Valine, leucine, isoleucine, phenylalanine and tyrosine levels (compare Fig. 2) were significantly elevated in obese subjects (9) and inversely correlated with immunoreactive insulin levels. The branch-chained amino acid levels are also significantly elevated in patients with diabetic acidosis (1 l). It has therefore been suggested (9) that leucine, which apparently induces insulin release by a different mechanism from that of glucose and arginine (8), acts as a physiological insulin feed-back regulator. In Fig. 3, the plasma valine level of small infants during breast-feeding is compared with that of newborn infants before feeding (7, 20, 24), and the plasma levels of infants on different amounts of cow-milk protein feeding (31). It is evident from this figure, that in order to reach a plasma valine level equivalent to that of breast-fed infants, the cow-milk based formula should be consumed in amounts of 1.5 g/kg. This is considerably lower than what is customary, but is comparable to the requirement for protein of normal full-term infants of no greater than 1.72 g/lOO kcal (1.15 g/lOO ml) found by Fomon et al. (12). The low cystine and taurine levels in formula-fed infants are consistent with the low cystine content of casein and non-humanized Acta Puediatr Scund 67
250
200
-
2oo
150
-
150
loo
130 -
50
~
ALA
GLY
SER
! U
Lys
VAL
THR
ORN
cys
TAU
LEU
HIS
ARG
TYR
GL~
ASN
PHE
ClTR
Fig. 2. Peripheral venous plasma freeamino acid concentration of 1 to 5-month-old human infants. Partly striped bars indicate breast-fed infants (mean +2 S.E., n=19), partly filled bars cow-milk formula-fed infants, 3-3.5 g protein/kg (meank2 S.E., n=29, ref. 31). The glutamine concentrations have been divided by 5 before being included in the diagram. The levels during formula-feeding of glutamine, glutamic acid and aspartic acid are of infants 1-2 years of age (ref. 33, using a lithium-buffer system identical to the one of the present investigation).
ASP ~ E T
5
cow-milk formula and the low amounts of taurine in cow-milk as compared to human milk (14, 15). This finding indicates a suboptimal supply of cystine and taurine. to cowmilk formula-fed infants, which could be important, as cystine is an essential amino acid, at least for prematurely born human infants (13). Besides, cystine is a precursor to taurine,
which seems to be essential and to play a major role in brain development (14, 34). The hypermethioninemia of excessive protein intake of infants, accidentally discovered during screening for inborn errors of amino acid metabolism in the U.S. (19) is not seen during 3-3.5 g/kg cow-milk protein feeding when compared with breast-feeding (Fig. 2). ACKNOWLEDGEMENT
200
, J
This investigation was supported by grants from the Swedish Medical Research Council No. 2583.
0 J
a
.
Y
150
-
-
REFERENCES
2
>
100
4
z
1.7
1.3 1
1.5
P R O T E I N INTAKE.
2
3 G/KG
BT
Fig. 3 . Peripheral venous plasma valine levels during early
infancy. The open bar indicates the result of the present investigation (breast-fed infants 1-5 months of age), the tilled bars those of Snyderman et al. (31) during different forms of cow-milk protein feeding; 1.3, 1.5, 1.7 and 3 g protein/kg in infants 1-3 months of age. The hatched area represents mean f 2 S.E. of newborn infants before the start of feeding procedures during the first day of life (24). The cross-hatched area represents mean k 2 S.E. of infants 1-5 years of age with the clinical syndrome of Kwashiorkor (protein deficiency) from 5 different developing countries (18). Acte Pwdicitr Scund 67
1. Abitbol, C. L., Feldman, D. B., Ahmann, P. & Rudman, D.: Plasma amino acid patterns during supplemental i.v. nutrition of low-birth-weight infants. J Pediatr, 86: 766, 1975. 2. Armstrong, M. D. & Stave, U.: A study of plasma free amino acid levels. Metabolism, 22: 549 and 571, 1973. 3. Benson, J. V . , Jr, Gordon, M. J. & Patterson, .I. A.: Accelerated chromatographic analysis of amino acids in physiological fluids containing glutamine and asparagine. Anal Biochem, 18: 228, 1967. 4. Bjornesjo, K. B.: The distribution of alpha amino nitrogen, urea nitrogen, and nonprotein nitrogen between erythrocytes and plasma in healthy males and females. Scand J Clin Lab Invest, 15: Suppl. 69, 25, 1963. 5 . Brodehl, J. & Gellissen, K.: Endogenous renal transport of free amino acids in infancy and childhood. Pediatrics, 42: 395, 1968. 6. Crofford, 0. B., Felts, P. W. & Lacy, W. W.: Effect of glucose infusion on the individual plasma free
Plasma amino acids in infants amino acids in man. Proc Soc Exp Biol Med, 117: 11, 1964. 7. Dickinson, J. C., Rosenbluhm, H. & Hamilton, P. B.: Ion exchange chromatography of the free amino acids in the plasma of the newborn infant. Pediatrics, 36: 2, 1965. 8. Fajans, S. S ., Quibrera, R., Pek, S . , Floyd, J. C., Jr, Christensen, H. N. & Conn, J. W.: Stimulation of insulin release in the dog by a non-metabolizable amino acid. Comparison with leucine and arginine. J Clin Endocrinol Metab, 33: 35, 1971. 9. Felig, P., Marliss, E. & Cahill, G. F., Jr: Plasma amino acid levels and insulin secretion in obesity. N E n g l J M e d , 2 8 1 : 8 1 1 , 1969. 10. Felig, P., Owen, 0. E., Wahren, J. & Cahill, F . G. Jr: Amino acid metabolism during prolonged starvation. J Clin Invest, 48: 584, 1969. 11. Felig, P., Marliss, E., Ohman, J. L. & Cahill, G. F., Jr: Plasma amino acid levels in diabetic ketoacidosis. Diabetes, 19: 727, 1970. 12. Fomon, S. J., Ziegler, E. E., Thomas, L. N. & Filer, L. J., Jr: Protein requirement of normal infants between 8 and 56 days of age. In J. H. P. Jonxis, H. K. A. Visser and J . A. Troelstra (eds.): Metabolic processes,in the foetus and newborn infant. Nutricia symposium. H. E. Stenfert Kroese N.V., Leiden 1971, p. 144. 13. Gaull, G., Sturman, J. A. & Raiha, N. C. R.: Development of mammalian sulfur metabolism: absence of cystathionase in human fetal tissues. Pediatr Res, 6: 538, 1972. 14. Gaull, G., Rassin, D. K., Raiha, N. R. C. & Heinonen, K.: Milk protein quantity and quality in lowbirth weight infants. 111. Effects on sulfur amino acids in plasma and urine. J Pediatr, 90: 348, 1977. IS. Ghadimi, H. & Pecora, P.: Free amino acids of different kinds of milk. Am J Clin Nutr, 13: 75, 1963. 16. Ghadimi, H. & Pecora, P.: Plasma amino acid levels after birth. Pediatrics, 34: 182, 1964. 17. Holmgren, G.: Effect of low, normal and high dietary protein intake on urinary amino acid excretion and plasma aminogram in children. Nutr Metab, 16: 223, 1974. 18. Holt, L. E., Jr, Snyderman, S. E., Norton, P. M., Roitman, E. & Finch, J.: The plasma aminogram in kwashiorkor. Lancet, 11: 7322, 1963. 19. Levy, H. L., Shih, V. E., Madigan, P. M., Karolkewicz, V., Cam, J. R., Lum, A., Richards, A . A,, Crawford, J. D. & MacCready, R. A.: Hypermethioninemia with other hyperaminoacidemias. Am J Dis Child, 117: 96, 1969. 20. Lindblad, B. S.: The venous plasma free amino acid levels during the first hours of life. I. After normal and short gestation and gestation complicated by hypertension. With special reference to the “small for dates” syndrome. Acta Paediatr Scand, 59: 13, 1970. 21. Lindblad, B. S . & Rahimtoola, R. J.: A pilot study on the quality of human milk in a lower socio-economic group of Karachi, Pakistan. Acta Paediatr Scand, 63: 125, 1974. 22. Lindblad, B . S., Settergren, G., Feychting, H. & Persson, B.: Total parenteral nutrition in infants.
’
23.
24.
25.
26.
27.
28. 29.
30.
31.
32. 33. 34.
35.
663
Blood levels of glucose, lactate, pyruvate, free fatty acids, glycerol, D-P-hydroxybutyrate, trig1ycerides, free amino acids and insulin. Acta Paediatr Scand, 66:409, 1977. Lindblad, B. S., Rahimtoola, R. J., Hafiz-ur-Rehman, Shamim Ahmad, S., Fancy, K., Singha, L. & Sajiad Hussain, S.: Plasma free amino acid levels during the initial rehabilitation of protein-energy malnutrition with protracted diarrhoea using a free amino acid-glucose diet. Acta Paediatr Scand. In press. Mestyan, J., Soltesz, Gy., Schultz, K. & Horvath, M.: Hyperaminoacidemia due to the accumulation of gluconeogenic amino acid precursors in hypoglycemic small-for-gestational age infants. J Pediatr, 87: 409, 1975. Nicolaidou, M., Lund, C. C. & McMenamy, R. H.: Unbound amino acid concentrations in plasma and erythrocytes of normal children and of cord blood. Arch Biochem Biophys, 96: 613, 1962. Posefsky, J., Felig, P., Tobin, J. D., Soeldner, J. S. & Cahill, G. F., Jr: Amino acid balance across tissues of the forearm in postabsorptive man. Effects of insulin at two dose levels. J Clin Invest, 48: 2273, 1969. Rassin, D. K., Gaull, G . E., Heinonen, K. & Raiha, N. R. C.: Milk protein quantity and quality in low birth weight infants. 11. Effects on selected aliphatic amino acids in plasma and urine. Pediatrics, 59: 407, 1977. Rigo, J. & Senterre, J.: Is taurine essential for the neonates? Biol Neonate, 32: 73, 1977. Saifer, A., Gerstenfeld, S. & Harris, A,: Photometric microdetermination of amino acids in biological fluids with the ninhydrin reaction. Clin Chim Acta, 5: 131, 1960. Scriver, C. R., Clow, C. L. & Lamm, P.: Plasma amino acids: screening, quantitation and interpretation. Am J Clin Nutr, 24: 876, 1971. Snyderman, S. E., Holt, L. E., Jr, Norton, P. M., Roitman, E. & Phansalkar, S . V.: The plasma aminogram. Influence of the level of protein intake and a comparison of whole protein and amino acid diets. PediatrRes, 2: 131, 1968. Spackman, D. H., Stein, W. H. & Moore, S.: Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem, 30: 1190, 1958. Stegink, L. D. & Baker, G. L.: Infusion of protein hydrolysates in the newborn infant: plasma amino acid concentrations. J Pediatr, 78: 595, 1971. Sturman, J . A., Rassin, D. K. & Gaull, G. E.: Taurine in developing rat brain: transfer of 35S-taurine to pups via the milk. Pediatr Res, 11: 28, 1977. Zinneman, H. H., Nuttall, F. Q. & Goetz, F. C.: Effect of endogenous insulin on human amino acid metabolism. Diabetes, 15: 5 , 1966.
Submitted Nov. 26, 1977 Accepted Febr. 22, 1978 (B. S. L.) Department of Paediatrics St. Goran’s Children’s Hospital Box 12500 S-112 81 Stockholm Sweden A< lo Pwdirrtr Sccrnd 67
PLASMA FREE AMINO ACID CONCENTRATIONS OF BREAST-FED INFANTS B. S. LINDBLAD, G . ALFVEN and R. ZETTERSTROM From the Department of Paediatrics, Karolinska Institutet, St Goran’s Children’s Hospital, Stockholm, Sweden
ABSTRACT. Lindblad, B. S., Alfven, G . and Zetterstrom, R. (Department of Paediatrics of Karolinska Institutet at St Goran’s Children’s Hospital, Stockholm, Sweden). Plasma free amino acid concentrations of breast-fed infants. Acta Paediatr Scand, 67: 659, 1978.Photometric determination of alpha-amino nitrogen in peripheral venous plasma and urine from 20 healthy, full-term infants, 1-5 months of age, showing normal growth and development during an uncomplicated lactation, revealed lower plasma levels than what has been found in adults, or 3.7k1.1 mg/100 ml, and a urinary excretion of 41k14 mg/24 hours. Ion-exchange chromatography of deproteinized peripheral venous plasma showed low valine concentrations, an increased glycine/valine ratio and high cystine and very high taurine levels when compared to the levels of healthy American infants of comparable ages fed 3-3.5 gtkg of cow-milk protein. The findings indicate that a formula based on cow-milk protein should optimally contain only 1.0-1.2 g protein/100 ml provided that it is “humanized” not only with regard to the lactalbumin/casein ratio, but also to the cystine and taurine content. The pattern of the plasma concentrations of free amino acids reported in the present investigation may be used as a normal reference for breast-fed infants. KEY WORDS: Amino acids, plasma, breast-feeding, human milk, cow-milk formula, valine, cystine, taurine, protein requirement
The free amino acid concentrations of plasma, as measured by ion-exchange chromatography, show little inter-individual variation if certain precautions are met with in the sampling, storage and analytical procedures (30). However, the plasma levels are dependent on age and sex (2) and on the protein intake (1, 16, 31). There are four investigations known to the authors where the plasma levels of free amino acids during childhood have been correlated to age and to dietary intake; two in premature infants (27, 28), one in full-term infants during formula feeding (31) and one in children 7-13 years of age (17). In view of the increasing need for amino acid analysis of plasma during screening, diagnosis and follow-up of inborn errors of amino acid metabolism (19), during the monitoring of total parenteral nutrition (22) and during the early diagnosis of nutritional inadequacy (23), there is obviously a need to establish the levels of breast-fed full-term infants, as opposed to
those of infants fed cow-milk formula with a considerably higher protein intake. The present investigation therefore aims at determining the post-alimentary free amino acid levels of deproteinized peripheral blood plasma in normal human infants, as represented by healthy full-term infants showing normal growth and development during an uncomplicated lactation. MATERIAL AND METHODS Twenty mothers, of a mean age of 24 years, who were selling their excess breast-milk to the Stockholm milk distribution centre were hospitalized with their healthy 1 to 5-month-old (mean 80-day old) infants. The mothers’ total milk volumes were 1244k381 m1/24 hours, according to test-weighing and mechanical pumping of excess milk at home before admission. The infants, 10 girls and 10 boys, were all full-term and showed a normal growth from normal birth weights and heights and were healthy with a normal development. They had all been fed ad libitum from the breast. Supported by a grant from the Swedish Medical Research Council (no. 2583). Aclu Paediutr Scand 67
660
B . S . Lindblad et al.
UMOL/L
PnOLlL
J5U
350
500
300
250
250
200
200
150
150
100
100
50
50
PRO RUI
LYS
SER
GLY
G U
LEU
CYS
THR
VAL
ORN
TAU
ASN
PIR
ARC
HIS
GLU
ILEU
ClTR
ME
Fig. 1. The free amino acid levels of peripheral venous blood plasma of 1 to S-monthold, breast-fed human infants. Mean ? S.D. are given.
ASP
MET
5
Blood was collected from the cubital vein in a heparinized tube before the early morning feed. The blood sample was centrifuged immediately; the plasma was deproteinized with crystalline sulphosalicylic acid, 50 mg/ ml, and the supernatant stored in -72°C until analyzed for alpha-amino nitrogen (29), and for individual free amino acids on a Bio-Cal 200 (Munchen) automatic amino acid analyser according to the method of Spackman et al. (32). A lithium buffer system (3) was used, allowing for the separation of serine, glutamine and asparagine. Tryptophan is lost during deproteinization and analysis, a s it is partly bound to albumin, and is not determined by this method. Cysteine was oxidized to cystine by allowing the sample to stand in room temperature at pH 7 for 4 hours. The methionine-sulphone peak was added to the one representing methionine during the estimation of the methionine concentration. Reproducibility was mean 4.9k0.5 (SE)% for the 23 parameters determined by ionexchange chromatography, ranging from 1.2 % (glutamine) to 9.4 7% (cystine). Urine was collected during a 24-hour period. The u r h e was kept in a refrigerator during collection and stored in -72°C until analyzed for alpha-amino nitrogen content (29). I
RESULTS /
The alpha-amino nitrogen concentration of plasma during uncomplicated lactation was foundtobe3.7k1.1 ( n = l l ) rng/lOOrnlandthe urinary excretion 41? 14 ( n=20) mg/24 hours. The individual free amino acid levels and the urea concentrations are given in Table 1. The results are also given in the form of an “arninogram” in Fig. 1 , which can serve as a Actu Puediutr Scund 67
reference chart on which to plot the results of plasma amino acid analyses during infancy. DISCUSSION The alpha-amino nitrogen concentration of plasma found in the present investigation is Table 1 . The plasma free amino acid and urea concentrations of breast-fed infants 1-5 months of age ~
~
Amino acid Taurine Aspartic acid Threonine Serine Glutamine Asparagine Glutamic acid Citrulline Proline Glycine Alanine 1/2 Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine Urea
pmol/l
S.D.
n
S.E.
90 15 108 1.56 663 43 63 23 195 187 269 98 113 21 4.5 89 49 31 102 119 84 83
34 7 50 56 208 18 27 13 90 66 96 39 47 8 16 23 25 15 38 61 22 29
19 19 19 19 19 18 19 6 19 19 19 19 18 18 19 19 19 17 16 19 19 16
8 2 11 13 48 4 6 5 21 15 22 9 I1 2 4 5 6 4 10 14 5 7
2 984
1613
19
375
Plasma amino acids in infants Table 2. Plasma valine concentrations during childhood Results which can be regarded as normal plasma levels for age are in italics
Investigation (reference) Newborn infants before feeding (24) Premature babies 1-2 weeks old (28) Human milk Formula 1.8 gll00 ml Formula 2.3 gll00 ml Premature babies less than 8 weeks old (27) 170 ml human milk/kg (800 pmol VAL/kg) Formula 1.5 g/lOO ml(l300 pmol VAL/kg) Formula 3 g/ I00 ml(2 500 pmol VAL/kg) 1-5 months ofage, human milk (present investigation) Ninth day of life, formula feeding (16) 1-3 months of age, formula feeding (3 1) 1.3 g proteinlkg 1.5 g proteinlkg 1.7 g protein/kg 3-3.5 g protein/kg 9 g protein/kg 4 days - 1 year of age (25) 4 days - 12 years of age (25) 4 months-2.5 years of age (16) 1-5 years of age, Kwashiorkor (18) 7-13 years of age (17) 0.21 g proteinikg 1.9g protein/kg
3.1 g protein/kg 6-18 years of age (2) Adults (2)
Result pmol/l, M f 2 S.E.
122f 10 139k34 219f36 274f44
120 180
280
113f22 247+38
91 111
167 194k 18 592 153f 6 171f 4 222k21 66f 6
194f55 212 +50 457+32 223f 7 252f 8
slightly lower than that reported for adults (4). Moreover, the individual amino acid levels of plasma SHOW considerably lower branchchained (valine, leucine, isoleucine) amino acid levels in comparison with those of older children and adults (2, 5 , 17, 25). The dependency on age of the valine plasma levels on the one hand, and on dietary protein intake on the other, is quite evident from the review of the literature summarized in Table 2. When the present results are compared with the plasma levels of free amino acids of “control” American infants of 1-3 months of age
661
being fed cow-milk based formula in amounts 3-3.5 g protein/kg in an investigation demonstrating the effect of overfeeding (9 g protein/ kg) on the plasma “aminogram” (31) it is clear that even the “controls” show the high valine levels and decreased glycinelvaline quotient characteristic of protein overnutrition (23, 3 1). This difference between normal breastfed Swedish young infants consuming 0.82 g protein/100 ml (21) and normal cow-milk formulafed American young infants consuming 2-2.3 g protein/100 ml (Fig. 2) could be of importance, as there are indications that the branchchained amino acid and tyrosine levels of plasma in particular influence insulin metabolism. The branch-chained amino acid levels of plasma seem to be especially sensitive to exogenous and endogenous insulin (6, 9, 10, 26, 35). Valine, leucine, isoleucine, phenylalanine and tyrosine levels (compare Fig. 2) were significantly elevated in obese subjects (9) and inversely correlated with immunoreactive insulin levels. The branch-chained amino acid levels are also significantly elevated in patients with diabetic acidosis (1 l). It has therefore been suggested (9) that leucine, which apparently induces insulin release by a different mechanism from that of glucose and arginine (8), acts as a physiological insulin feed-back regulator. In Fig. 3, the plasma valine level of small infants during breast-feeding is compared with that of newborn infants before feeding (7, 20, 24), and the plasma levels of infants on different amounts of cow-milk protein feeding (31). It is evident from this figure, that in order to reach a plasma valine level equivalent to that of breast-fed infants, the cow-milk based formula should be consumed in amounts of 1.5 g/kg. This is considerably lower than what is customary, but is comparable to the requirement for protein of normal full-term infants of no greater than 1.72 g/lOO kcal (1.15 g/lOO ml) found by Fomon et al. (12). The low cystine and taurine levels in formula-fed infants are consistent with the low cystine content of casein and non-humanized Acta Puediatr Scund 67
250
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Lys
VAL
THR
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ARG
TYR
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Fig. 2. Peripheral venous plasma freeamino acid concentration of 1 to 5-month-old human infants. Partly striped bars indicate breast-fed infants (mean +2 S.E., n=19), partly filled bars cow-milk formula-fed infants, 3-3.5 g protein/kg (meank2 S.E., n=29, ref. 31). The glutamine concentrations have been divided by 5 before being included in the diagram. The levels during formula-feeding of glutamine, glutamic acid and aspartic acid are of infants 1-2 years of age (ref. 33, using a lithium-buffer system identical to the one of the present investigation).
ASP ~ E T
5
cow-milk formula and the low amounts of taurine in cow-milk as compared to human milk (14, 15). This finding indicates a suboptimal supply of cystine and taurine. to cowmilk formula-fed infants, which could be important, as cystine is an essential amino acid, at least for prematurely born human infants (13). Besides, cystine is a precursor to taurine,
which seems to be essential and to play a major role in brain development (14, 34). The hypermethioninemia of excessive protein intake of infants, accidentally discovered during screening for inborn errors of amino acid metabolism in the U.S. (19) is not seen during 3-3.5 g/kg cow-milk protein feeding when compared with breast-feeding (Fig. 2). ACKNOWLEDGEMENT
200
, J
This investigation was supported by grants from the Swedish Medical Research Council No. 2583.
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-
REFERENCES
2
>
100
4
z
1.7
1.3 1
1.5
P R O T E I N INTAKE.
2
3 G/KG
BT
Fig. 3 . Peripheral venous plasma valine levels during early
infancy. The open bar indicates the result of the present investigation (breast-fed infants 1-5 months of age), the tilled bars those of Snyderman et al. (31) during different forms of cow-milk protein feeding; 1.3, 1.5, 1.7 and 3 g protein/kg in infants 1-3 months of age. The hatched area represents mean f 2 S.E. of newborn infants before the start of feeding procedures during the first day of life (24). The cross-hatched area represents mean k 2 S.E. of infants 1-5 years of age with the clinical syndrome of Kwashiorkor (protein deficiency) from 5 different developing countries (18). Acte Pwdicitr Scund 67
1. Abitbol, C. L., Feldman, D. B., Ahmann, P. & Rudman, D.: Plasma amino acid patterns during supplemental i.v. nutrition of low-birth-weight infants. J Pediatr, 86: 766, 1975. 2. Armstrong, M. D. & Stave, U.: A study of plasma free amino acid levels. Metabolism, 22: 549 and 571, 1973. 3. Benson, J. V . , Jr, Gordon, M. J. & Patterson, .I. A.: Accelerated chromatographic analysis of amino acids in physiological fluids containing glutamine and asparagine. Anal Biochem, 18: 228, 1967. 4. Bjornesjo, K. B.: The distribution of alpha amino nitrogen, urea nitrogen, and nonprotein nitrogen between erythrocytes and plasma in healthy males and females. Scand J Clin Lab Invest, 15: Suppl. 69, 25, 1963. 5 . Brodehl, J. & Gellissen, K.: Endogenous renal transport of free amino acids in infancy and childhood. Pediatrics, 42: 395, 1968. 6. Crofford, 0. B., Felts, P. W. & Lacy, W. W.: Effect of glucose infusion on the individual plasma free
Plasma amino acids in infants amino acids in man. Proc Soc Exp Biol Med, 117: 11, 1964. 7. Dickinson, J. C., Rosenbluhm, H. & Hamilton, P. B.: Ion exchange chromatography of the free amino acids in the plasma of the newborn infant. Pediatrics, 36: 2, 1965. 8. Fajans, S. S ., Quibrera, R., Pek, S . , Floyd, J. C., Jr, Christensen, H. N. & Conn, J. W.: Stimulation of insulin release in the dog by a non-metabolizable amino acid. Comparison with leucine and arginine. J Clin Endocrinol Metab, 33: 35, 1971. 9. Felig, P., Marliss, E. & Cahill, G. F., Jr: Plasma amino acid levels and insulin secretion in obesity. N E n g l J M e d , 2 8 1 : 8 1 1 , 1969. 10. Felig, P., Owen, 0. E., Wahren, J. & Cahill, F . G. Jr: Amino acid metabolism during prolonged starvation. J Clin Invest, 48: 584, 1969. 11. Felig, P., Marliss, E., Ohman, J. L. & Cahill, G. F., Jr: Plasma amino acid levels in diabetic ketoacidosis. Diabetes, 19: 727, 1970. 12. Fomon, S. J., Ziegler, E. E., Thomas, L. N. & Filer, L. J., Jr: Protein requirement of normal infants between 8 and 56 days of age. In J. H. P. Jonxis, H. K. A. Visser and J . A. Troelstra (eds.): Metabolic processes,in the foetus and newborn infant. Nutricia symposium. H. E. Stenfert Kroese N.V., Leiden 1971, p. 144. 13. Gaull, G., Sturman, J. A. & Raiha, N. C. R.: Development of mammalian sulfur metabolism: absence of cystathionase in human fetal tissues. Pediatr Res, 6: 538, 1972. 14. Gaull, G., Rassin, D. K., Raiha, N. R. C. & Heinonen, K.: Milk protein quantity and quality in lowbirth weight infants. 111. Effects on sulfur amino acids in plasma and urine. J Pediatr, 90: 348, 1977. IS. Ghadimi, H. & Pecora, P.: Free amino acids of different kinds of milk. Am J Clin Nutr, 13: 75, 1963. 16. Ghadimi, H. & Pecora, P.: Plasma amino acid levels after birth. Pediatrics, 34: 182, 1964. 17. Holmgren, G.: Effect of low, normal and high dietary protein intake on urinary amino acid excretion and plasma aminogram in children. Nutr Metab, 16: 223, 1974. 18. Holt, L. E., Jr, Snyderman, S. E., Norton, P. M., Roitman, E. & Finch, J.: The plasma aminogram in kwashiorkor. Lancet, 11: 7322, 1963. 19. Levy, H. L., Shih, V. E., Madigan, P. M., Karolkewicz, V., Cam, J. R., Lum, A., Richards, A . A,, Crawford, J. D. & MacCready, R. A.: Hypermethioninemia with other hyperaminoacidemias. Am J Dis Child, 117: 96, 1969. 20. Lindblad, B. S.: The venous plasma free amino acid levels during the first hours of life. I. After normal and short gestation and gestation complicated by hypertension. With special reference to the “small for dates” syndrome. Acta Paediatr Scand, 59: 13, 1970. 21. Lindblad, B. S . & Rahimtoola, R. J.: A pilot study on the quality of human milk in a lower socio-economic group of Karachi, Pakistan. Acta Paediatr Scand, 63: 125, 1974. 22. Lindblad, B . S., Settergren, G., Feychting, H. & Persson, B.: Total parenteral nutrition in infants.
’
23.
24.
25.
26.
27.
28. 29.
30.
31.
32. 33. 34.
35.
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Blood levels of glucose, lactate, pyruvate, free fatty acids, glycerol, D-P-hydroxybutyrate, trig1ycerides, free amino acids and insulin. Acta Paediatr Scand, 66:409, 1977. Lindblad, B. S., Rahimtoola, R. J., Hafiz-ur-Rehman, Shamim Ahmad, S., Fancy, K., Singha, L. & Sajiad Hussain, S.: Plasma free amino acid levels during the initial rehabilitation of protein-energy malnutrition with protracted diarrhoea using a free amino acid-glucose diet. Acta Paediatr Scand. In press. Mestyan, J., Soltesz, Gy., Schultz, K. & Horvath, M.: Hyperaminoacidemia due to the accumulation of gluconeogenic amino acid precursors in hypoglycemic small-for-gestational age infants. J Pediatr, 87: 409, 1975. Nicolaidou, M., Lund, C. C. & McMenamy, R. H.: Unbound amino acid concentrations in plasma and erythrocytes of normal children and of cord blood. Arch Biochem Biophys, 96: 613, 1962. Posefsky, J., Felig, P., Tobin, J. D., Soeldner, J. S. & Cahill, G. F., Jr: Amino acid balance across tissues of the forearm in postabsorptive man. Effects of insulin at two dose levels. J Clin Invest, 48: 2273, 1969. Rassin, D. K., Gaull, G . E., Heinonen, K. & Raiha, N. R. C.: Milk protein quantity and quality in low birth weight infants. 11. Effects on selected aliphatic amino acids in plasma and urine. Pediatrics, 59: 407, 1977. Rigo, J. & Senterre, J.: Is taurine essential for the neonates? Biol Neonate, 32: 73, 1977. Saifer, A., Gerstenfeld, S. & Harris, A,: Photometric microdetermination of amino acids in biological fluids with the ninhydrin reaction. Clin Chim Acta, 5: 131, 1960. Scriver, C. R., Clow, C. L. & Lamm, P.: Plasma amino acids: screening, quantitation and interpretation. Am J Clin Nutr, 24: 876, 1971. Snyderman, S. E., Holt, L. E., Jr, Norton, P. M., Roitman, E. & Phansalkar, S . V.: The plasma aminogram. Influence of the level of protein intake and a comparison of whole protein and amino acid diets. PediatrRes, 2: 131, 1968. Spackman, D. H., Stein, W. H. & Moore, S.: Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem, 30: 1190, 1958. Stegink, L. D. & Baker, G. L.: Infusion of protein hydrolysates in the newborn infant: plasma amino acid concentrations. J Pediatr, 78: 595, 1971. Sturman, J . A., Rassin, D. K. & Gaull, G. E.: Taurine in developing rat brain: transfer of 35S-taurine to pups via the milk. Pediatr Res, 11: 28, 1977. Zinneman, H. H., Nuttall, F. Q. & Goetz, F. C.: Effect of endogenous insulin on human amino acid metabolism. Diabetes, 15: 5 , 1966.
Submitted Nov. 26, 1977 Accepted Febr. 22, 1978 (B. S. L.) Department of Paediatrics St. Goran’s Children’s Hospital Box 12500 S-112 81 Stockholm Sweden A< lo Pwdirrtr Sccrnd 67
