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Infusion of protein hydrolysates in the newborn infant: Plasma amino acid concentrations
April, 1971 T h e Journal o/ P E D I A T R I C S
595
Infusion of protein bydrolysates in doe newborn infant: Plasma amino acid concentrations Protein hydrolysate infusions commonly administered to young infants unable to sustain adequate protein intake contain large amounts of glutamate and aspartate. Neurotoxic effects have been reported in other species and were attributed to high doses of these amino acids. Plasma glutamate and aspartate levels in infants treated with such preparations were within normal limits, but other amino acids were markedly below fasting levels. Plasma amino acid levels quickly reflected the amino acid composition of the hydrolysate and some degree of amino acid imbalance resulted when either of the 2 products employed in this study constituted the sole source of protein intake.
Lewis D, Stegink, Ph.D., and George L. Baker, M.D. IOWA CIITY~ IOWA
ADMINISTRATION Of highcalorie solutions by central venous catheter to infants who are unable to sustain an adequate oral intake has recently become popular. 1-~ Infants benefiting from such therapy include those severely ill and debilitated with intractable diarrhea from a variety of causes or those with surgically correctable congenital malformations of the gastrointestinal tract. The intravenous preparations supply adequate calories in the form of dextrose and sufficient quantities of a protein hydrolysate to achieve a positive nitrogen balance and net protein synthesis. Despite common use of such preparations, no studies THE
From the Departments of Pediatrics and Biochemistry, The University of Iowa College of Medicine. Supported in part by Grants-In-Aid from the Gerber Products Company and the Jimmy Durante Children's Research Fund.
are available to our knowledge concerning the effect of such infusions on plasma amino acid levels. In addition, a number of investigators have reported acute and irreversible degenerative changes in the infant mouse retina following subcutaneous injection of either monosodium glutamate or aspartate# -9 Adult mice were much more resistant to glutamateinduced lesions than newborn animals, 4 and glutamate injection of pregnant mice produced no observable abnormalities in the offspring# Olney and Sharpe 9-11 have reported that the arcuate nucleus of the hypothalamus is particularly vulnerable to monosodium glutamate injected subcutaneously at dose levels ranging from 0.5 to 2 Gin. per kilog-ram of body weight in the infant mouse, rat, rabbit, and a single immature rhesus monkey; Adamo and Rather 12 were unable Vol. 78, No. 4, pp. 595-602
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to reproduce such effects in the infant rat. Olney and Ho is recently reported that oral ingestion of high levels of glutamate, aspartate, or cysteine by the infant mouse also produces such lesions. McLaughlan and associates 14 reported plasma glutamate levels were increased 3 to 3.5 times and aspartate levels doubled at peak absorption (20 minutes) after ingestion of 0.2 Gm. of monosodium glutamate per kilogram by the young rat who had been fasted overnight. A slower rise in glutamate levels was noted when the glutamate was administered in the presence of food proteins; 30 minutes after administration, plasma glutamate was 3 times normal values and still increasing. Brain glutamate concentrations were not affected in these animals. Similar results have been reported by Schwerin and associates15 for the mouse. However, subcutaneous injection of glutamate bypasses 2 important organs controlling plasma glutamate levels: the liver and the gut. Neame and Wiseman ~6,17 and Matthews and Wiseman ~s have shown that substantial quantities of ingested glutamate are absorbed as alanine. The alanine is undoubtedly formed by glutamate dependent transamination of pyruvate (produced either by glycolysis, or from glutamate itself via a-ketoglutarate, succinyl-coenzyme A, fumarate and malate by Kreb's cycle enzymes, and conversion of malate to pyruvate via nicotinamide adenine dinueleotide phosphate [NADP +] malate dehydrogenase). Our studies with the newborn pig indicate that the normal high levels of glutamate in the portal blood (48 ~moles per 100 ml.) are drastically reduced in peripheral blood (8 /zmoles per 100 ml.) upon passage through the liver even in absence of glutamate load. 19 Large amounts of glutamate and aspartate, normally found at low levels in the plasma, are contained in the 2 most commonly used intravenous preparations: Amigen and Aminosol. In view of tbe reported neurotoxic effects of monosodium glutamate in the newborn animal, the use of protein hydrolysate infusions to treat young infants is of considerable concern. Infants on such therapy usually
The ]ournal o[ Pediatrics April 1971
receive 120 ml. of hydrolysate per kilogram per 24 hours by constant infusion. This amounts to 288 rag. of glutamate and 73 rag. of aspartate in casein hydrolysates and 80 rag. of glutamate and 105 mg. of aspartate in beef fibrin hydrolysates per kilogram per day. Since these preparations are infused into the superior vena cava bypassing hepatic and gut control, and in view of the reported neurotoxic effects of injected glutamate and aspartate, it is imperative to know what effect, if any, such infusions have on plasma glutamate and aspartate concentrations. METHODS
Heparinized blood samples from patients and normal control infants, drawn at 2 v.xI. to avoid circadian rhythm influences, were immediately centrifuged to remove the erythrocytes and deproteinized with sulfosalacylic acid. 2~ The deproteinized plasma was either analyzed immediately or stored at -80 ~ C. until assay (never more than 24 hours) to minimize conversion of glutamine to glutamie acid and pyrrolidone carboxylic acid and avoid the loss of cystine.TM 25 Amino acid analyses were carried out on Technicon NC-1 amino acid analyzers using the buffer system described by Efron 2~ and the automatic temperature control system developed in this laboratory. 23 The free amino acid compositipn of the protein hydrolysate soluti0ns ~Was: determined after treating 2 ml. of the solution with 100 rag. solid sulfosalacylic acid. Replicate aliquotes were analyzed at 3 concentrations (0.01 ml., 0.05 ml., and 0.10 ml.) to obtain accurate estimations of amino acids present in both large and small amounts~ The hydrolysate preparations studied were Amigen, (Baxter Lab., Morton Grove, Ill.) and Aminosol (Abbott Lab., North Chicago, Ill.) Patients treated with these protein hydrolysate infusions receive 120 ml. of hydrolysate per kilogram per 24 hours by constant infusion via the superior vena cava. These protein hydrolysates differ in their source of protein; Amigen is prepared from casein and Aminosol from beef fibrin. The amino acid
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Protein hydrolysate infusion in neonate
Table I. Free amino acid composition of protein hydrolysate preparations for infusion Amigen
Amino acid Taurine Cysteic acid Hydroxyproline Methionine sulfoxides Aspartic acid Threonine Serine Asparagine Glutamine Glutamie acid Proline Citrulline Glycine Alanine Valine 1/2 Cystlne Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine 1-Methylhistidine Histidine Arginine Tryptophan
Amlnosol
(casein) (b eel fibrin) (mieromoIes/ l O0 ml.) Not detected Not detected Not detected 112 Not detected Not detected 117 15 500 922 1,900 Not detected Not detected 1,960 1,280 5 758 1,109 1,686 Not detected 754 1,365 2,960 234 1,182 40 2,493 Not detected 631 165 165
660 860 1,398 Not detected Not detected 358 1,020 7 5,630 1,150 404 75 476 580 3,950 348 381 80 1,190 32 326 690 110
Table II. Composition of protein hydrolysate infusion (content per liter)
Infusion Protein Glucose Lipid Sodium Potassium Chloride Phosphate Calcium Magnesium Folic acid Ascorbic acid Vitamin A Vitamin D Thiamine I-ICI Riboflavin Niacinamide Pyridoxine HC1 Panthenol Vitamin E Heparin sodium
Amount 25 Gin. 225 Gin. 30 20 20 15 500 50 37 500 10,000 1,000 50 10 100 15 25 5 1,000
mEq. mEq. mEq. mEq. mEq. mg. mg. mg. U.S.P. units U.S.P. units mg. mg. mg. mg. mg. International Units U.
597
composition of these 2 preparations determined in our laboratory by replicate analysis at several concentrations is shown in Table I. T h e free amino acid content of the products analyzed in our laboratory varied considerably from the composition stated on the label and also differ from each other. Both preparations differ, considerably in composition from plasma amino acid levels and contain large amounts of glutamate and aspartate, normally found at low levels in plasma. Intravenous lipid preparations are not available for clinical use at this time, thus the solutions commonly used do not contain a source of lipid. In order to supply essential fatty acids, a plasma infusion is given each week. Calories, minerals, electrolytes, and vitamins are supplied in adequate amounts (Table I I ) . Because of high solute concentration these solutions must be given into a vessel with high blood flow, and the superior vena cava is most frequently chosen. Eight plasma free amino acid determinations from 6 infants receiving such preparations by central venous catheter were carried out. Three infants were debilitated because of severe chronic diarrhea, one was debilitated with recurrent postoperative bowel fistulae, one had giant cell hepatitis with hypoglycemia, and one was well nourished but required surgical repair of a large omphalocele. Six samples Of plasma were drawn while the infants were receiving a casein hydrolysate and 2 while a beef fibrin hydrolysate was being infused. The duration of hydrolysate administration before plasma amino acid analysis was performed ranged from 2 to 54 days. Patient K. C. was receiving a casein hydrolysate orally in addition to the parenteral feeding. Patient B. D. was studied with each of the protein hydrolysate preparations. RESULTS
Plasma amino acid levels in infants treated with these preparations are shown in Table I I I , along with normal plasma amino acid levels for 1 month old infants , 2 hours postprandially, and for 1- to 2-year-old children
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Table III. Plasma amino acid levels in young infants treated with protein Plasma amino acid levels Patients C. E.
Age 5o Diagnosis: ! Chronic dlarrhea IDa: 2 Amino IPt: Casein acid iN~: Debilitated Taurine 2.22 Hydroxyproline 0.30 Aspartate 1.70 Threonine. 11.9 Serine 12.5 Glutamine 47.7 Glutamate 9.40 Proline 24.4 Citrulline 1.94 Glycine 22.8 Alanine 17.5 a-Aminobutyrate 0.60 Valine 20.3 1/2-Cystine 1.67 Methionlne 3.05 Isoleucine 7.50 Leueine 10.8 Tyroslne 1.40 Phenylalanine 8.05 Ornithine 4.15 Lysine 14.8 Histidine 6.39 Arginine 3.33
Age (day~): 13 Diagnosis: Chronic I diarrhea D: 2 P: Casein IN: Debilitated 3.05 0.40 1.67 6.94 15.6 25.5 7.78 11.4 0.83 20.3 13.9 0.65 9.99 1.94 1.95 4.44 8.05 1.45 5.30 3.61 15.0 7.22 1.40
K.C.
C.P.
R.S.
Age (days): 68 Diagnosis: Hepatitis D: 28 P: Casein + oral N: Fair
Age (days) : 92 Diagnosis: Chronic diarrhea D: 54 P: Casein N: Debilitated
Age (days): 150 Diagnosis: Fistulae D: 6 P: Casein N: Debilitated
0.60 t .71 1.43 15.6 8.03 36.9 1.34 9.36 1.21 16.0 22.5 0.70 9.48 2.34 1.89 2.72 6.54 4.00 3.90 6.00 17.1 3.23 2.36
1.40 0.90 0.62 11.2 11.2 38.5 5.18 12.2 1.69 24.8 21.1 0.53 13.4 1.37 2.95 4.91 9.45 3.68 9.06 5.77 15.0 5.50 3.78
1.00 1.64 0.42 15.4 18.6 41.5 8.00 16.9 1.47 33.9 35.0 0.60 10.3 5.18 2.55 2.80 4.41 2.99 6.67 4.50 9.70 5.96 2.55
eDays on preparation. "~Protein based of infusion. +*Nutritional status.
after an overnight fast, as determined in this laboratory. Plasma glutamate and aspartate levels were within normal nonfasting limits in all infants studied. Substantial conversion of infused glutamate and aspartate to glutamine and asparagine, respectively, was apparent, since these latter amino acids are not found in the hydrolysate preparations. In our experience, infants treated with these 2 preparations remain in relative positive nitrogen balance, grow, and show no recognizable neurotoxic symptoms. Infant K. C. was known to have giant cell hepatitis. Despite a moderate to severe degree of liver damage demonstrated by hypoglycemia, abnormaI Iiver function tests, and biopsy, plasma levels
of glutamate and aspartate were within normal limits. It is difficult to imagine glutamate-induced neural damage without substantial elevation of plasma glutamate levels at least of the order noted by McLaughlan and associates. 14 Certain plasma amino acid concentrations in these patients were, however, well below normal fasting levels observed in full-term infants. The plasma amino acid levels closely reflected the amino acid composition of the protein hydrolysate. Infants infused with casein hydrolysates had subfasting plasma concentrations of hydroxyproline, valine, arginine, tyrosine, and cystine. In contrast to normal plasma aminograms where phenylalanine to tyrosine ratios are near unity, the
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h y d r o l y s a t e infusions a n d in n o r m a l subjects (mieromoles per 100 ml. plasma) B. O.
Age (days) : 6 Diagnosis: Omphalocele
D: 3 P: Fibrin N: Good 2.75 0.30 0.20 20.3 11.5 45.3 3.25 11.8 1.25 30.5 7.50 0.15 3.00 4.00 2.25 3.00 7.50 1.50 2.12 2.50 9.00 4.00 4.00
B. O.
Normal subjects N~15 N:15
B. D.
Age (days): 12 Age (days): 10 Diagnosis: Omphalo- Diagnosis: Omphalocele cele D:2 D: 7 P: Casein P: Fibrin N: Good N: Good 12.8 3.10 1.25 2.30 0.63 0.53 36.0 23.2 19.3 13.2 57.3 47.8 5.75 5.01 15.0 22.9 0.88 0.77 57.0 27.8 10.8 7.94 0.43 0.17 4.30 12.9 6.00 3.14 4.00 2.64 5.25 4.62 12.5 7.00 2.75 2.39 2.75 6.01 4.76 2.14 14.8 9.29 5.75 5.28 6.00 3.21
ratio of p l a s m a p h e n y l a l a n i n e to tyrosine rem a i n e d at the elevated ratio f o u n d in the hydrolysate ( 5 : 1 ) . T h e one exception to this unusual ratio was I n f a n t C. P. This infant h a d been treated with the casein hydrolysate infusion for a considerable length of time, a n d i n d u c t i o n of enzymes necessary to h a n dle b o t h p h e n y l a l a n i n e a n d g l u t a m a t e m a y have occurred since the p h e n y l a l a n i n e to tyrosine ratios were normM, a n d g l u t a m a t e was at the fasting level. I n d u c t i o n a n d suppression of enzymes to h a n d l e increased levels of a m i n o acids would be consistent with d a t a presented by Daniel a n d W a i s m a n 24 after long-term feeding of excess dietary m e t h i o nine in the rat. P l a s m a cystine levels are p a r t i c u l a r l y low in infants fed casein hydroly-
Age (mo.): 1 Diagnosis: Normal D: -P" Normal diet N: 2 hr. postprandial 12.2 • 4.22 9.99 + 2.47 2.12 + 2.27 20.4 +- 3.43 23.6 -+ 5.34 70.3 -+ 14.6 7.70 • 2.53 33.6 • 6.75 4.23 -+ 1.91 34.8 • 5.26 40.9 + 7.17 2.46 +- 2.71 34.6 -+ 7.06 9.93 + 3.00 4.91 -+ 1.45 13.9 + 3.29 21.8 • 4.96 12.6 -+ 3.40 11.2 • 1.59 13.9 + 5.76 24.3 - 4.66 10.4 + 2.35 12.9 • 4.12
Age (yr,) : 1-2 Diagnosis: Normal D:-P: Normal diet N: Overnight last 10.3 + 4.14 5.36 + 2.06 0.47 • 0.44 12.1 -+ 4.15 14.3 + 2.02 54.9 + 3.71 2.73 • 1.53 16.5 + 6.46 3.20 -+ 3.28 23.0 + 5.40 27.7 -2_ 6.26 1.70 -+- 2.80 24.5 • 4.74 9.45 • 2.72 2.72 • 1.71 7.62 -+ 2.41 13.2 +- 2.09 6.91 + 1.80 6.12 -+ 1.80 5.81 +- 2.21 16.3 +- 2.56 8.18 • 1.34 7.48 • 1.52
sate p r e p a r a t i o n s , reflecting the absence of this a m i n o acid in the hydrolysate, a factor of some concern in the i m m a t u r e infant (see Discussion). T h e one exception was I n f a n t C. P. who was receiving some oral intake. C e r t a i n p l a s m a a m i n o acid levels were also below fasting in I n f a n t B. D., who was t r e a t e d with the beef fibrin hydrolysate p r e p aration. A l t h o u g h p l a s m a cysfine a n d arginine levels m o r e closely resembled n o r m a l (when c o m p a r e d to levels in infants t r e a t e d with casein hydrolysate p r e p a r a t i o n s ) , hydroxyproline, valine, a n d tyrosine were below fasting levels with both p r e p a r a t i o n s ; alanine a n d p h e n y l a l a n i n e values were especially low in this infant, a l t h o u g h the phenyla l a n i n e to tyrosine ratio a p p r o a c h e d unity.
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DISCUSSION
Although concern about the composition of protein hydrolysate preparations, particularly in regard to glutamate toxicity, is reasonable, our data indicate that the young infant is able to adequately control plasma glutamate levels at the infusion rates used in this study. We have followed several patients from our center for 18 months or longer postinfusion and have been unable to detect any demonstrable evidence of central nervous system damage or growth change. Although the side effect of nausea and vomiting occuring with the use of such infusions is usually attributed to monosodium glutamate levels, Vinnars and associates~5 report the occurrence of these symptoms in patients infused with Intramin Novum which contains no glutamate. Our data demonstrate that the amino acid composition of the protein hydrolysate preparations directly affect plasma amino acid levels, and indicate that each preparation produces a characteristic plasma amino acid pattern. A comparison of the amino acid composition of the 2 preparations studied (Table I) demonstrates that the plasma amino acid changes which occur are logical. Beef fibrin hydrolysates contain equal molar quantities of phenylalanine and tyrosine, accounting for the 1:1 ratio found in the plasma of infants treated with such preparations, but contain a lower absolute amount of phenylalanine when compared to the casein hydrolysate preparation, resulting in decreased plasma phenylalanine levels. The considerably lower valine content of the beef fibrin preparation is reflected in the lower plasma levels of this amino acid. Since the beef fibrin hydrolysate contains larger quantities of glycine, arginine, and cystine than the casein hydrolysate, plasma levels of these amino acids would be expected to be higher in the infant treated with this preparation. A comparison of Patient B.D.'s plasma amino acid levels after a change from the beef fibrin to the casein hydrolysate preparations (10 and 12 days of age) support this concept. Glycine, cystine, arginine, and threonine levels were decreased, valine
The Journal o/ Pediatrics April 1971
and phenylalanine levels increased, and the phenylalanine to tyrosine ratio was changed from unity to 5 : 2. The reason for the lower alanine levels in Infant B. D. is not clear. Both preparations contain approximately equal amounts of this amino acid, and no substantial change in alanine levels occurred when the infant was shifted from the beef fibrin to the casein hydrolysate preparation. A priori, it would seem that the amino acid content of the protein infusion should resemble that of normal human plasma as closely as possible, since the infusion enters directly into circulation without passage through the gastrointestinal tract and liver. Although considerable care has been taken to supplement these preparations with adequate amounts of "essential amino acids," little concern appears to have been given to matching plasma amino acid levels. The terms "essential" and "nonessential" amino acid relate only to dietary requirements when oral feeding is used, and have no meaning with respect to the relative importance of each amino acid metabolically. We feel that serious consideration should be given to possible subacute states of amino acid imbalance, antagonism or toxicity resulting from the present formulation of these products. The basis for the adverse effects attributed to these conditions has not been established, but a number of investigators have reported striking changes in plasma amino acid levels under conditions of dietary amino acid imbalance (see review in reference 26). Studies in experimental animals indicate that the homeostatic response to an altered balance of amino acids in the blood and body fluids seems to inhibit the animal from ingesting an excessive amount of a severely unbalanced diet. Forced feedings of such a severely unbalanced diet have resulted in degeneration of certain organs and even in early deathY' 2s The absence or low levels of cystine or cysteine in these preparations is of special concern in the treatment of the premature and full-term newborn infant. Although cysteine is readily formed from methionine
Volume 78 Number 4
via cystathionine in the h u m a n adult, Sturm a n a n d associates 29 have recently p r o v i d e d strong evidence t h a t cysteine (or cystine) m a y be an essential amino acid in the h u m a n fetus, p r e m a t u r e , a n d full-term n e w b o r n infant. I n view of the low p l a s m a cysteine values noted in these studies, especially with Amigen, it a p p e a r s that some s u p p l e m e n t a tion with cysteine or cystine is desirable. O u r d a t a indicate t h a t the amino acid composition of the hydrolysate has a p r o found effect on p l a s m a a m i n o acid levels a n d suggest a degree of a m i n o acid i m b a l a n c e in b o t h products. Most of the studies which have defined the conditions of a m i n o acid imbalance, toxicity, or antagonism have utilized diets administered by way of the gastrointestinal tract, although some studies of a m i n o acid toxicity have utilized the injection of amino acid supplied in excess. This a n d the p a u c i t y of d a t a in the literature concerning the effects of intravenous feedings on p l a s m a a m i n o acid levels m a k e it difficult to generalize directly as to the extent of imb a l a n c e present in o u r patients. I t seems obvious t h a t a better physiologic response of the p a t i e n t could be achieved if such p r e p a rations were f o r m u l a t e d to m o r e closely m a t c h p l a s m a a m i n o acid levels. Studies evaluating such p r e p a r a t i o n s are currently in progress.
REFERENCES
1. Dudrick, S. J., Wilmore, D. W., Vars, H. M., and Rhoads, J. E.: Long-term total parenteral nutrition with growth, development, and positive nitrogen balance, Surgery 64: 134, 1968. 2. Wilmore, D. W., and Dudrick, S. J.: Growth and development of an infant receiving all nutrients exclusively by vein, J. A. M. A. 203: 860, 1968. 3. Filler, R. M., Eraklis, A. J., Rubin, V. G., and Das, J. B.: Long-term total parenteral nutrition in infants, New Eng. J. Med. 381: 589, 1969. 4. Lucas, D. R., and Newhouse, J. P.: The toxic effect of sodium L-glutamate on the inner layers of the retina, Arch. Ophthal. 58: 193, 1957. 5. Potts, A. M., Modrell, K. W., and Kingsbury, C.: Permanent fractionation of the electroretinogram by sodium glutamate, Amer. J. Ophthal. 50: 900, 1960.
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6. Freedman, J. K., and Potts, A. M.: Repression of glutaminase I in rat retina by administration of sodium L-glutamate, Invest. Ophthal. 1: 118, 1962. 7. Freedman, J. K., and Potts, A. IV[.: Repression of glutaminase I in the rat retina by administration of sodium L-glutamate II, Invest. Ophthal. 2: 252, 1967. 8. Cohen, A. I.: An electron microscopic study of the modification by monosodium glutamate on the retinas of normal and "rodless" mice, Amer. J. Anat. 120: 319, 1967. 9. Olney, J. W.: Glutamate induced retinal degeneration in neonatal mice. Electron microscopy of the acutely evolving lesion, J. Neuropath. Exp. Neurol. 98: 455, 1969. 10. Olney, J. W.: Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate, Science 164: 719, 1969. 11. Olney, J. W., and Sharpe, L. G.: Brain lesions in an infant rhesus monkey treated with monosodium glutamate, Science 166: 386, 1969. 12. Adamo, N. J., and Ratner, A.: Monosodium glutamate: Lack of effects on brain and reproductive function in rats, Science 169: 673, 1970. 13. Olney, J. W., and Ho, O. L.: Brain damage in infant mice following oral intake of glutamate, aspartate or cysteine, Nature 227: 609, 1970. 14. McLaughlan, J. M., Noel, F. J., Botting, H. G., and Knipfel, J. E.: Blood and brain levels of glutamic acid in young rats given monosodium glutamate, Nutr. Rep. Internat. 1: 131, 1970. 15. Schwerln, P., Bessman, S. P., and Waelsch, H.: The uptake of glutamic acid and glutamine by brain and other tissues of the rat and mouse, J. Biol. Chem. 184: 37, 1950. 16. Neame, K. D., and Wiseman, G.: The transamination of glutamic and asparatie acids during absorption by the small intestine of the dog in vivo, J. Physiol. 135: 442, 1957. 17. Neame, K. D., and Wiseman, G.: The alanine and oxo acid concentrations in mesenteric blood during the absorption of L-glutamic acid by the small intestine of the dog, cat and rabbit in vivo, J. Physiol. 140: 148, 1958. 18. Matthews, D. M., and Wiseman, G.: Transamination by the small intestine of the rat, J. Physiol. 120: 55P, 1953. 19. Stegink, L. D., Baker, G. L., and Filer, L. J., Jr.: Unpublished data. 20. Efron, M. L.: Quantitative estimation of amino acids in physiological fluids using a Technicon amino acid analyzer, in Skeggs, L. T., Jr., editor: Automation in analytical chemistry, New York, 1966, Mediad Inc., p. 637. 21. Perry, T. L., and Hansen, S.: Technical pitfalls leading to errors in the quantitation of plasma amino acids, Clln. Chim. Acta 25: 53, 1969. 22. Dickinson, J. C., Rosenblum, H., and Hamilton, P. B.: Ion exchange chromatography of
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24. 25.
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free amino acids in the plasma of the newborn infant, Pediatrics 36: 2, 1965. Meyer, P. D., Steglnk, L. D., and Shlpton, H. W.: Two multitemperature bath control units for single column amino acid analyzers, J. Chromatogr. 48: 538, 1970. Daniel, R. G., and Waisman, H. A.: Adaptation of the weanling rat to diets containing excess methionine, J. Nutr. 99: 299, 1969. Vinnars, E., Furst, P., Hermansson, I. L., Josephson, B., and Lindolmer, B.: Protein catabolism in the postoperative state and its treatment with amino acid solution, Acta Chir. 8cand, 136: 95, 1970. Harper, A. E.: Amino acid toxlcitles and imbalance, in Munro, H. N., and Allison, J. B., editors: Mammalian protein metabolism, Vol.
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2, New York, 1964, Academic Press, Inc., p. 87. 27. Sidransky, H., and Baba, T.: Chemical pathology of acute amino acid deficiencies IIL Morphologlc and biochemlcal changes in young rats fed valine- or lysine-devoid diets~ J. Nutr. 70: 463, 1960. 28. Sidransky, H., and Rechcigl, M., Jr.: Chemical pathology of acute amino acid deficiencies V. Comparison of morphologlc and biochemical changes in young rats fed proteinfree or thronine-free diets, J. Nutr. 78: 269, 1962. 29. Sturman, J. A., Gaull, G., and Raiha, N. C. R.: Absence of cystathionase in human fetal liver: Is cystine essential? Science 169: 74, 1970.
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Infusion of protein bydrolysates in doe newborn infant: Plasma amino acid concentrations Protein hydrolysate infusions commonly administered to young infants unable to sustain adequate protein intake contain large amounts of glutamate and aspartate. Neurotoxic effects have been reported in other species and were attributed to high doses of these amino acids. Plasma glutamate and aspartate levels in infants treated with such preparations were within normal limits, but other amino acids were markedly below fasting levels. Plasma amino acid levels quickly reflected the amino acid composition of the hydrolysate and some degree of amino acid imbalance resulted when either of the 2 products employed in this study constituted the sole source of protein intake.
Lewis D, Stegink, Ph.D., and George L. Baker, M.D. IOWA CIITY~ IOWA
ADMINISTRATION Of highcalorie solutions by central venous catheter to infants who are unable to sustain an adequate oral intake has recently become popular. 1-~ Infants benefiting from such therapy include those severely ill and debilitated with intractable diarrhea from a variety of causes or those with surgically correctable congenital malformations of the gastrointestinal tract. The intravenous preparations supply adequate calories in the form of dextrose and sufficient quantities of a protein hydrolysate to achieve a positive nitrogen balance and net protein synthesis. Despite common use of such preparations, no studies THE
From the Departments of Pediatrics and Biochemistry, The University of Iowa College of Medicine. Supported in part by Grants-In-Aid from the Gerber Products Company and the Jimmy Durante Children's Research Fund.
are available to our knowledge concerning the effect of such infusions on plasma amino acid levels. In addition, a number of investigators have reported acute and irreversible degenerative changes in the infant mouse retina following subcutaneous injection of either monosodium glutamate or aspartate# -9 Adult mice were much more resistant to glutamateinduced lesions than newborn animals, 4 and glutamate injection of pregnant mice produced no observable abnormalities in the offspring# Olney and Sharpe 9-11 have reported that the arcuate nucleus of the hypothalamus is particularly vulnerable to monosodium glutamate injected subcutaneously at dose levels ranging from 0.5 to 2 Gin. per kilog-ram of body weight in the infant mouse, rat, rabbit, and a single immature rhesus monkey; Adamo and Rather 12 were unable Vol. 78, No. 4, pp. 595-602
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to reproduce such effects in the infant rat. Olney and Ho is recently reported that oral ingestion of high levels of glutamate, aspartate, or cysteine by the infant mouse also produces such lesions. McLaughlan and associates 14 reported plasma glutamate levels were increased 3 to 3.5 times and aspartate levels doubled at peak absorption (20 minutes) after ingestion of 0.2 Gm. of monosodium glutamate per kilogram by the young rat who had been fasted overnight. A slower rise in glutamate levels was noted when the glutamate was administered in the presence of food proteins; 30 minutes after administration, plasma glutamate was 3 times normal values and still increasing. Brain glutamate concentrations were not affected in these animals. Similar results have been reported by Schwerin and associates15 for the mouse. However, subcutaneous injection of glutamate bypasses 2 important organs controlling plasma glutamate levels: the liver and the gut. Neame and Wiseman ~6,17 and Matthews and Wiseman ~s have shown that substantial quantities of ingested glutamate are absorbed as alanine. The alanine is undoubtedly formed by glutamate dependent transamination of pyruvate (produced either by glycolysis, or from glutamate itself via a-ketoglutarate, succinyl-coenzyme A, fumarate and malate by Kreb's cycle enzymes, and conversion of malate to pyruvate via nicotinamide adenine dinueleotide phosphate [NADP +] malate dehydrogenase). Our studies with the newborn pig indicate that the normal high levels of glutamate in the portal blood (48 ~moles per 100 ml.) are drastically reduced in peripheral blood (8 /zmoles per 100 ml.) upon passage through the liver even in absence of glutamate load. 19 Large amounts of glutamate and aspartate, normally found at low levels in the plasma, are contained in the 2 most commonly used intravenous preparations: Amigen and Aminosol. In view of tbe reported neurotoxic effects of monosodium glutamate in the newborn animal, the use of protein hydrolysate infusions to treat young infants is of considerable concern. Infants on such therapy usually
The ]ournal o[ Pediatrics April 1971
receive 120 ml. of hydrolysate per kilogram per 24 hours by constant infusion. This amounts to 288 rag. of glutamate and 73 rag. of aspartate in casein hydrolysates and 80 rag. of glutamate and 105 mg. of aspartate in beef fibrin hydrolysates per kilogram per day. Since these preparations are infused into the superior vena cava bypassing hepatic and gut control, and in view of the reported neurotoxic effects of injected glutamate and aspartate, it is imperative to know what effect, if any, such infusions have on plasma glutamate and aspartate concentrations. METHODS
Heparinized blood samples from patients and normal control infants, drawn at 2 v.xI. to avoid circadian rhythm influences, were immediately centrifuged to remove the erythrocytes and deproteinized with sulfosalacylic acid. 2~ The deproteinized plasma was either analyzed immediately or stored at -80 ~ C. until assay (never more than 24 hours) to minimize conversion of glutamine to glutamie acid and pyrrolidone carboxylic acid and avoid the loss of cystine.TM 25 Amino acid analyses were carried out on Technicon NC-1 amino acid analyzers using the buffer system described by Efron 2~ and the automatic temperature control system developed in this laboratory. 23 The free amino acid compositipn of the protein hydrolysate soluti0ns ~Was: determined after treating 2 ml. of the solution with 100 rag. solid sulfosalacylic acid. Replicate aliquotes were analyzed at 3 concentrations (0.01 ml., 0.05 ml., and 0.10 ml.) to obtain accurate estimations of amino acids present in both large and small amounts~ The hydrolysate preparations studied were Amigen, (Baxter Lab., Morton Grove, Ill.) and Aminosol (Abbott Lab., North Chicago, Ill.) Patients treated with these protein hydrolysate infusions receive 120 ml. of hydrolysate per kilogram per 24 hours by constant infusion via the superior vena cava. These protein hydrolysates differ in their source of protein; Amigen is prepared from casein and Aminosol from beef fibrin. The amino acid
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Protein hydrolysate infusion in neonate
Table I. Free amino acid composition of protein hydrolysate preparations for infusion Amigen
Amino acid Taurine Cysteic acid Hydroxyproline Methionine sulfoxides Aspartic acid Threonine Serine Asparagine Glutamine Glutamie acid Proline Citrulline Glycine Alanine Valine 1/2 Cystlne Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine 1-Methylhistidine Histidine Arginine Tryptophan
Amlnosol
(casein) (b eel fibrin) (mieromoIes/ l O0 ml.) Not detected Not detected Not detected 112 Not detected Not detected 117 15 500 922 1,900 Not detected Not detected 1,960 1,280 5 758 1,109 1,686 Not detected 754 1,365 2,960 234 1,182 40 2,493 Not detected 631 165 165
660 860 1,398 Not detected Not detected 358 1,020 7 5,630 1,150 404 75 476 580 3,950 348 381 80 1,190 32 326 690 110
Table II. Composition of protein hydrolysate infusion (content per liter)
Infusion Protein Glucose Lipid Sodium Potassium Chloride Phosphate Calcium Magnesium Folic acid Ascorbic acid Vitamin A Vitamin D Thiamine I-ICI Riboflavin Niacinamide Pyridoxine HC1 Panthenol Vitamin E Heparin sodium
Amount 25 Gin. 225 Gin. 30 20 20 15 500 50 37 500 10,000 1,000 50 10 100 15 25 5 1,000
mEq. mEq. mEq. mEq. mEq. mg. mg. mg. U.S.P. units U.S.P. units mg. mg. mg. mg. mg. International Units U.
597
composition of these 2 preparations determined in our laboratory by replicate analysis at several concentrations is shown in Table I. T h e free amino acid content of the products analyzed in our laboratory varied considerably from the composition stated on the label and also differ from each other. Both preparations differ, considerably in composition from plasma amino acid levels and contain large amounts of glutamate and aspartate, normally found at low levels in plasma. Intravenous lipid preparations are not available for clinical use at this time, thus the solutions commonly used do not contain a source of lipid. In order to supply essential fatty acids, a plasma infusion is given each week. Calories, minerals, electrolytes, and vitamins are supplied in adequate amounts (Table I I ) . Because of high solute concentration these solutions must be given into a vessel with high blood flow, and the superior vena cava is most frequently chosen. Eight plasma free amino acid determinations from 6 infants receiving such preparations by central venous catheter were carried out. Three infants were debilitated because of severe chronic diarrhea, one was debilitated with recurrent postoperative bowel fistulae, one had giant cell hepatitis with hypoglycemia, and one was well nourished but required surgical repair of a large omphalocele. Six samples Of plasma were drawn while the infants were receiving a casein hydrolysate and 2 while a beef fibrin hydrolysate was being infused. The duration of hydrolysate administration before plasma amino acid analysis was performed ranged from 2 to 54 days. Patient K. C. was receiving a casein hydrolysate orally in addition to the parenteral feeding. Patient B. D. was studied with each of the protein hydrolysate preparations. RESULTS
Plasma amino acid levels in infants treated with these preparations are shown in Table I I I , along with normal plasma amino acid levels for 1 month old infants , 2 hours postprandially, and for 1- to 2-year-old children
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Table III. Plasma amino acid levels in young infants treated with protein Plasma amino acid levels Patients C. E.
Age 5o Diagnosis: ! Chronic dlarrhea IDa: 2 Amino IPt: Casein acid iN~: Debilitated Taurine 2.22 Hydroxyproline 0.30 Aspartate 1.70 Threonine. 11.9 Serine 12.5 Glutamine 47.7 Glutamate 9.40 Proline 24.4 Citrulline 1.94 Glycine 22.8 Alanine 17.5 a-Aminobutyrate 0.60 Valine 20.3 1/2-Cystine 1.67 Methionlne 3.05 Isoleucine 7.50 Leueine 10.8 Tyroslne 1.40 Phenylalanine 8.05 Ornithine 4.15 Lysine 14.8 Histidine 6.39 Arginine 3.33
Age (day~): 13 Diagnosis: Chronic I diarrhea D: 2 P: Casein IN: Debilitated 3.05 0.40 1.67 6.94 15.6 25.5 7.78 11.4 0.83 20.3 13.9 0.65 9.99 1.94 1.95 4.44 8.05 1.45 5.30 3.61 15.0 7.22 1.40
K.C.
C.P.
R.S.
Age (days): 68 Diagnosis: Hepatitis D: 28 P: Casein + oral N: Fair
Age (days) : 92 Diagnosis: Chronic diarrhea D: 54 P: Casein N: Debilitated
Age (days): 150 Diagnosis: Fistulae D: 6 P: Casein N: Debilitated
0.60 t .71 1.43 15.6 8.03 36.9 1.34 9.36 1.21 16.0 22.5 0.70 9.48 2.34 1.89 2.72 6.54 4.00 3.90 6.00 17.1 3.23 2.36
1.40 0.90 0.62 11.2 11.2 38.5 5.18 12.2 1.69 24.8 21.1 0.53 13.4 1.37 2.95 4.91 9.45 3.68 9.06 5.77 15.0 5.50 3.78
1.00 1.64 0.42 15.4 18.6 41.5 8.00 16.9 1.47 33.9 35.0 0.60 10.3 5.18 2.55 2.80 4.41 2.99 6.67 4.50 9.70 5.96 2.55
eDays on preparation. "~Protein based of infusion. +*Nutritional status.
after an overnight fast, as determined in this laboratory. Plasma glutamate and aspartate levels were within normal nonfasting limits in all infants studied. Substantial conversion of infused glutamate and aspartate to glutamine and asparagine, respectively, was apparent, since these latter amino acids are not found in the hydrolysate preparations. In our experience, infants treated with these 2 preparations remain in relative positive nitrogen balance, grow, and show no recognizable neurotoxic symptoms. Infant K. C. was known to have giant cell hepatitis. Despite a moderate to severe degree of liver damage demonstrated by hypoglycemia, abnormaI Iiver function tests, and biopsy, plasma levels
of glutamate and aspartate were within normal limits. It is difficult to imagine glutamate-induced neural damage without substantial elevation of plasma glutamate levels at least of the order noted by McLaughlan and associates. 14 Certain plasma amino acid concentrations in these patients were, however, well below normal fasting levels observed in full-term infants. The plasma amino acid levels closely reflected the amino acid composition of the protein hydrolysate. Infants infused with casein hydrolysates had subfasting plasma concentrations of hydroxyproline, valine, arginine, tyrosine, and cystine. In contrast to normal plasma aminograms where phenylalanine to tyrosine ratios are near unity, the
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599
h y d r o l y s a t e infusions a n d in n o r m a l subjects (mieromoles per 100 ml. plasma) B. O.
Age (days) : 6 Diagnosis: Omphalocele
D: 3 P: Fibrin N: Good 2.75 0.30 0.20 20.3 11.5 45.3 3.25 11.8 1.25 30.5 7.50 0.15 3.00 4.00 2.25 3.00 7.50 1.50 2.12 2.50 9.00 4.00 4.00
B. O.
Normal subjects N~15 N:15
B. D.
Age (days): 12 Age (days): 10 Diagnosis: Omphalo- Diagnosis: Omphalocele cele D:2 D: 7 P: Casein P: Fibrin N: Good N: Good 12.8 3.10 1.25 2.30 0.63 0.53 36.0 23.2 19.3 13.2 57.3 47.8 5.75 5.01 15.0 22.9 0.88 0.77 57.0 27.8 10.8 7.94 0.43 0.17 4.30 12.9 6.00 3.14 4.00 2.64 5.25 4.62 12.5 7.00 2.75 2.39 2.75 6.01 4.76 2.14 14.8 9.29 5.75 5.28 6.00 3.21
ratio of p l a s m a p h e n y l a l a n i n e to tyrosine rem a i n e d at the elevated ratio f o u n d in the hydrolysate ( 5 : 1 ) . T h e one exception to this unusual ratio was I n f a n t C. P. This infant h a d been treated with the casein hydrolysate infusion for a considerable length of time, a n d i n d u c t i o n of enzymes necessary to h a n dle b o t h p h e n y l a l a n i n e a n d g l u t a m a t e m a y have occurred since the p h e n y l a l a n i n e to tyrosine ratios were normM, a n d g l u t a m a t e was at the fasting level. I n d u c t i o n a n d suppression of enzymes to h a n d l e increased levels of a m i n o acids would be consistent with d a t a presented by Daniel a n d W a i s m a n 24 after long-term feeding of excess dietary m e t h i o nine in the rat. P l a s m a cystine levels are p a r t i c u l a r l y low in infants fed casein hydroly-
Age (mo.): 1 Diagnosis: Normal D: -P" Normal diet N: 2 hr. postprandial 12.2 • 4.22 9.99 + 2.47 2.12 + 2.27 20.4 +- 3.43 23.6 -+ 5.34 70.3 -+ 14.6 7.70 • 2.53 33.6 • 6.75 4.23 -+ 1.91 34.8 • 5.26 40.9 + 7.17 2.46 +- 2.71 34.6 -+ 7.06 9.93 + 3.00 4.91 -+ 1.45 13.9 + 3.29 21.8 • 4.96 12.6 -+ 3.40 11.2 • 1.59 13.9 + 5.76 24.3 - 4.66 10.4 + 2.35 12.9 • 4.12
Age (yr,) : 1-2 Diagnosis: Normal D:-P: Normal diet N: Overnight last 10.3 + 4.14 5.36 + 2.06 0.47 • 0.44 12.1 -+ 4.15 14.3 + 2.02 54.9 + 3.71 2.73 • 1.53 16.5 + 6.46 3.20 -+ 3.28 23.0 + 5.40 27.7 -2_ 6.26 1.70 -+- 2.80 24.5 • 4.74 9.45 • 2.72 2.72 • 1.71 7.62 -+ 2.41 13.2 +- 2.09 6.91 + 1.80 6.12 -+ 1.80 5.81 +- 2.21 16.3 +- 2.56 8.18 • 1.34 7.48 • 1.52
sate p r e p a r a t i o n s , reflecting the absence of this a m i n o acid in the hydrolysate, a factor of some concern in the i m m a t u r e infant (see Discussion). T h e one exception was I n f a n t C. P. who was receiving some oral intake. C e r t a i n p l a s m a a m i n o acid levels were also below fasting in I n f a n t B. D., who was t r e a t e d with the beef fibrin hydrolysate p r e p aration. A l t h o u g h p l a s m a cysfine a n d arginine levels m o r e closely resembled n o r m a l (when c o m p a r e d to levels in infants t r e a t e d with casein hydrolysate p r e p a r a t i o n s ) , hydroxyproline, valine, a n d tyrosine were below fasting levels with both p r e p a r a t i o n s ; alanine a n d p h e n y l a l a n i n e values were especially low in this infant, a l t h o u g h the phenyla l a n i n e to tyrosine ratio a p p r o a c h e d unity.
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DISCUSSION
Although concern about the composition of protein hydrolysate preparations, particularly in regard to glutamate toxicity, is reasonable, our data indicate that the young infant is able to adequately control plasma glutamate levels at the infusion rates used in this study. We have followed several patients from our center for 18 months or longer postinfusion and have been unable to detect any demonstrable evidence of central nervous system damage or growth change. Although the side effect of nausea and vomiting occuring with the use of such infusions is usually attributed to monosodium glutamate levels, Vinnars and associates~5 report the occurrence of these symptoms in patients infused with Intramin Novum which contains no glutamate. Our data demonstrate that the amino acid composition of the protein hydrolysate preparations directly affect plasma amino acid levels, and indicate that each preparation produces a characteristic plasma amino acid pattern. A comparison of the amino acid composition of the 2 preparations studied (Table I) demonstrates that the plasma amino acid changes which occur are logical. Beef fibrin hydrolysates contain equal molar quantities of phenylalanine and tyrosine, accounting for the 1:1 ratio found in the plasma of infants treated with such preparations, but contain a lower absolute amount of phenylalanine when compared to the casein hydrolysate preparation, resulting in decreased plasma phenylalanine levels. The considerably lower valine content of the beef fibrin preparation is reflected in the lower plasma levels of this amino acid. Since the beef fibrin hydrolysate contains larger quantities of glycine, arginine, and cystine than the casein hydrolysate, plasma levels of these amino acids would be expected to be higher in the infant treated with this preparation. A comparison of Patient B.D.'s plasma amino acid levels after a change from the beef fibrin to the casein hydrolysate preparations (10 and 12 days of age) support this concept. Glycine, cystine, arginine, and threonine levels were decreased, valine
The Journal o/ Pediatrics April 1971
and phenylalanine levels increased, and the phenylalanine to tyrosine ratio was changed from unity to 5 : 2. The reason for the lower alanine levels in Infant B. D. is not clear. Both preparations contain approximately equal amounts of this amino acid, and no substantial change in alanine levels occurred when the infant was shifted from the beef fibrin to the casein hydrolysate preparation. A priori, it would seem that the amino acid content of the protein infusion should resemble that of normal human plasma as closely as possible, since the infusion enters directly into circulation without passage through the gastrointestinal tract and liver. Although considerable care has been taken to supplement these preparations with adequate amounts of "essential amino acids," little concern appears to have been given to matching plasma amino acid levels. The terms "essential" and "nonessential" amino acid relate only to dietary requirements when oral feeding is used, and have no meaning with respect to the relative importance of each amino acid metabolically. We feel that serious consideration should be given to possible subacute states of amino acid imbalance, antagonism or toxicity resulting from the present formulation of these products. The basis for the adverse effects attributed to these conditions has not been established, but a number of investigators have reported striking changes in plasma amino acid levels under conditions of dietary amino acid imbalance (see review in reference 26). Studies in experimental animals indicate that the homeostatic response to an altered balance of amino acids in the blood and body fluids seems to inhibit the animal from ingesting an excessive amount of a severely unbalanced diet. Forced feedings of such a severely unbalanced diet have resulted in degeneration of certain organs and even in early deathY' 2s The absence or low levels of cystine or cysteine in these preparations is of special concern in the treatment of the premature and full-term newborn infant. Although cysteine is readily formed from methionine
Volume 78 Number 4
via cystathionine in the h u m a n adult, Sturm a n a n d associates 29 have recently p r o v i d e d strong evidence t h a t cysteine (or cystine) m a y be an essential amino acid in the h u m a n fetus, p r e m a t u r e , a n d full-term n e w b o r n infant. I n view of the low p l a s m a cysteine values noted in these studies, especially with Amigen, it a p p e a r s that some s u p p l e m e n t a tion with cysteine or cystine is desirable. O u r d a t a indicate t h a t the amino acid composition of the hydrolysate has a p r o found effect on p l a s m a a m i n o acid levels a n d suggest a degree of a m i n o acid i m b a l a n c e in b o t h products. Most of the studies which have defined the conditions of a m i n o acid imbalance, toxicity, or antagonism have utilized diets administered by way of the gastrointestinal tract, although some studies of a m i n o acid toxicity have utilized the injection of amino acid supplied in excess. This a n d the p a u c i t y of d a t a in the literature concerning the effects of intravenous feedings on p l a s m a a m i n o acid levels m a k e it difficult to generalize directly as to the extent of imb a l a n c e present in o u r patients. I t seems obvious t h a t a better physiologic response of the p a t i e n t could be achieved if such p r e p a rations were f o r m u l a t e d to m o r e closely m a t c h p l a s m a a m i n o acid levels. Studies evaluating such p r e p a r a t i o n s are currently in progress.
REFERENCES
1. Dudrick, S. J., Wilmore, D. W., Vars, H. M., and Rhoads, J. E.: Long-term total parenteral nutrition with growth, development, and positive nitrogen balance, Surgery 64: 134, 1968. 2. Wilmore, D. W., and Dudrick, S. J.: Growth and development of an infant receiving all nutrients exclusively by vein, J. A. M. A. 203: 860, 1968. 3. Filler, R. M., Eraklis, A. J., Rubin, V. G., and Das, J. B.: Long-term total parenteral nutrition in infants, New Eng. J. Med. 381: 589, 1969. 4. Lucas, D. R., and Newhouse, J. P.: The toxic effect of sodium L-glutamate on the inner layers of the retina, Arch. Ophthal. 58: 193, 1957. 5. Potts, A. M., Modrell, K. W., and Kingsbury, C.: Permanent fractionation of the electroretinogram by sodium glutamate, Amer. J. Ophthal. 50: 900, 1960.
Protein hydrolysate in/usion in neonate
60 1
6. Freedman, J. K., and Potts, A. M.: Repression of glutaminase I in rat retina by administration of sodium L-glutamate, Invest. Ophthal. 1: 118, 1962. 7. Freedman, J. K., and Potts, A. IV[.: Repression of glutaminase I in the rat retina by administration of sodium L-glutamate II, Invest. Ophthal. 2: 252, 1967. 8. Cohen, A. I.: An electron microscopic study of the modification by monosodium glutamate on the retinas of normal and "rodless" mice, Amer. J. Anat. 120: 319, 1967. 9. Olney, J. W.: Glutamate induced retinal degeneration in neonatal mice. Electron microscopy of the acutely evolving lesion, J. Neuropath. Exp. Neurol. 98: 455, 1969. 10. Olney, J. W.: Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate, Science 164: 719, 1969. 11. Olney, J. W., and Sharpe, L. G.: Brain lesions in an infant rhesus monkey treated with monosodium glutamate, Science 166: 386, 1969. 12. Adamo, N. J., and Ratner, A.: Monosodium glutamate: Lack of effects on brain and reproductive function in rats, Science 169: 673, 1970. 13. Olney, J. W., and Ho, O. L.: Brain damage in infant mice following oral intake of glutamate, aspartate or cysteine, Nature 227: 609, 1970. 14. McLaughlan, J. M., Noel, F. J., Botting, H. G., and Knipfel, J. E.: Blood and brain levels of glutamic acid in young rats given monosodium glutamate, Nutr. Rep. Internat. 1: 131, 1970. 15. Schwerln, P., Bessman, S. P., and Waelsch, H.: The uptake of glutamic acid and glutamine by brain and other tissues of the rat and mouse, J. Biol. Chem. 184: 37, 1950. 16. Neame, K. D., and Wiseman, G.: The transamination of glutamic and asparatie acids during absorption by the small intestine of the dog in vivo, J. Physiol. 135: 442, 1957. 17. Neame, K. D., and Wiseman, G.: The alanine and oxo acid concentrations in mesenteric blood during the absorption of L-glutamic acid by the small intestine of the dog, cat and rabbit in vivo, J. Physiol. 140: 148, 1958. 18. Matthews, D. M., and Wiseman, G.: Transamination by the small intestine of the rat, J. Physiol. 120: 55P, 1953. 19. Stegink, L. D., Baker, G. L., and Filer, L. J., Jr.: Unpublished data. 20. Efron, M. L.: Quantitative estimation of amino acids in physiological fluids using a Technicon amino acid analyzer, in Skeggs, L. T., Jr., editor: Automation in analytical chemistry, New York, 1966, Mediad Inc., p. 637. 21. Perry, T. L., and Hansen, S.: Technical pitfalls leading to errors in the quantitation of plasma amino acids, Clln. Chim. Acta 25: 53, 1969. 22. Dickinson, J. C., Rosenblum, H., and Hamilton, P. B.: Ion exchange chromatography of
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24. 25.
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free amino acids in the plasma of the newborn infant, Pediatrics 36: 2, 1965. Meyer, P. D., Steglnk, L. D., and Shlpton, H. W.: Two multitemperature bath control units for single column amino acid analyzers, J. Chromatogr. 48: 538, 1970. Daniel, R. G., and Waisman, H. A.: Adaptation of the weanling rat to diets containing excess methionine, J. Nutr. 99: 299, 1969. Vinnars, E., Furst, P., Hermansson, I. L., Josephson, B., and Lindolmer, B.: Protein catabolism in the postoperative state and its treatment with amino acid solution, Acta Chir. 8cand, 136: 95, 1970. Harper, A. E.: Amino acid toxlcitles and imbalance, in Munro, H. N., and Allison, J. B., editors: Mammalian protein metabolism, Vol.
The ]ournal o[ Pediatrics April 1971
2, New York, 1964, Academic Press, Inc., p. 87. 27. Sidransky, H., and Baba, T.: Chemical pathology of acute amino acid deficiencies IIL Morphologlc and biochemlcal changes in young rats fed valine- or lysine-devoid diets~ J. Nutr. 70: 463, 1960. 28. Sidransky, H., and Rechcigl, M., Jr.: Chemical pathology of acute amino acid deficiencies V. Comparison of morphologlc and biochemical changes in young rats fed proteinfree or thronine-free diets, J. Nutr. 78: 269, 1962. 29. Sturman, J. A., Gaull, G., and Raiha, N. C. R.: Absence of cystathionase in human fetal liver: Is cystine essential? Science 169: 74, 1970.