1Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, T6G 2P5; 2The Research Institute, The Hospital for Sick Children, and the Departments of 3Paediatrics and 4Nutritional Science, University of Toronto, Toronto, Ontario, Canada M5G 1X8
Submitted 28 July 2003 ; accepted in final form 11 May 2004
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ABSTRACT |
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proline; glutamate; arginine requirement; biosynthesis; amino acid kinetics; first-pass metabolism
In a previous study (7), we demonstrated that feeding arginine-free diets to neonatal piglets led to severe life-threatening hyperammonemia in hours. In that study, we also determined that proline, but not glutamate, was an effective precursor in ameliorating arginine deficiency-induced hyperammonemia. In preliminary studies, we were able to demonstrate that the gastrically fed piglet could be fed arginine at a minimum level of 0.20 g·kg1·day1 with only a moderate, but steady, increase in plasma ammonia over 5 days (unpublished observations). We hypothesized that this level of arginine intake would lead to a maximal rate of endogenous arginine synthesis over days. The arginine requirement for piglets has been estimated at 0.40 g·kg1·day1 by the National Research Council (NRC) (22), and we have generally employed a standard diet with 0.90 g·kg1·day1 without compromising growth or nitrogen balance. As a result, we fed arginine at a generous level of 1.80 g·kg1·day1 to determine the basal or obligatory rate of endogenous arginine synthesis.
The mechanisms governing the rate of endogenous arginine synthesis in the neonate are not known. Studies in a mature rodent model suggest that renal arginine synthesis is independent of dietary arginine intake (14). Similarly, isotopic investigations conducted in healthy men fed arginine-adequate or arginine-free diets suggest that de novo arginine synthesis is not increased in response to a reduced level of dietary arginine (10). Arginine homeostasis in the adult human, therefore, appears to be maintained via altering arginine degradation relative to dietary intake and metabolic state. This hypothesis is supported by a significantly reduced rate of conversion of arginine to ornithine measured in the plasma, combined with a 46% reduction in ornithine oxidation in healthy men fed arginine-free diets (11). Because the major site of arginine synthesis differs between the adult (kidney) and the neonate (small intestine) (35), the regulation of arginine synthesis should be examined separately in the neonate. Furthermore, to the authors' knowledge, the relative contributions made by proline and glutamate to arginine synthesis have never been examined under different dietary intakes of arginine in the neonate.
The present study was designed to elucidate whether the neonatal intestine has a basal and a maximal arginine synthetic capacity, and whether the dietary arginine intake affects the degree to which proline and glutamate are used as precursors for arginine synthesis.
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MATERIALS AND METHODS |
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Piglets were initially anesthetized with an intramuscular injection of acepromazine (0.5 mg/kg Atravet; Ayerst Laboratories, Montreal, QC, Canada) and ketamine hydrochloride (10 mg/kg Rogarsetic; Rogar STB, Montreal, QC, Canada) and subsequently maintained under a mixture of oxygen and halothane (Fluothane; Ayerst Laboratories, Montreal, QC, Canada) throughout surgery (day 0). Three Silastic catheters (Ed-Art, Don Mills, ON, Canada) were installed into the stomach, femoral vein, and umbilical vein by aseptic technique (37). A Stamm gastrostomy was performed in all piglets to install the gastric catheter, which was used for intragastric (IG) feeding and tracer infusion. The blood sampling catheter was introduced into the femoral vein and advanced to the inferior vena cava just caudal to the heart. The umbilical catheter was inserted transperitoneally into the umbilical vein and guided to the portal-hepatic junction. Radiolabeled tracers were infused into this catheter, thereby isolating the effects of first-pass gut metabolism (4). Catheters were tunneled under the skin from the point of exit on the left side of the chest to the points of entry into the blood vessels or stomach. To ensure the patency of the umbilical catheter, a 0.9% saline solution was infused continuously at 1 ml/h throughout the study period. Immediately following surgery, piglets were given injections of buprenorphine hydrochloride (0.05 mg Buprenex; Reckitt and Colman Pharmaceutical, Richmond, VA), trimethoprim (10 mg) and sulfadoxine (50 mg Borgal; Hoechst Roussel Vet Canada, Regina, SK, Canada), and gentamicin sulfate (5 mg Garasol; Schering-Plough Animal Health, Pointe-Claire, QC, Canada). Gentamicin sulfate, trimethoprim, and sulfadoxine injections were continued for 6 days following surgery.
Animals were housed individually in circular wire mesh cages that were cleaned daily. Cages were arranged in banks of four, allowing visual and auditory contact with other piglets. Toys were provided for environmental enrichment. The room was lit from 0800 to 2000, and the temperature was maintained at 28°C, with supplemental heat provided by heat lamps.
Diets. An elemental and complete diet was infused continuously through a tether-swivel system (Alice King Chatham Medical Arts, Los Angeles, CA) using pressure-sensitive infusion pumps. The infusion regimen was designed to supply all nutrients piglets require (37) when administered at targeted intakes of 15 g amino acids·kg1·day1 and 1.1 MJ metabolizable energy·kg1·day1, with glucose and lipid each supplying 50% of nonprotein energy. The amino acid composition of the complete diet was similar to that of an elemental total parenteral nutrition solution that is based on human milk protein (Vaminolact; Fresenius Kabi, Uppsala, Sweden) and consisted of (in mg/g total L-amino acids) 106 alanine, 61 arginine, 61 aspartate, 16 cysteine, 105 glutamate, 27 glycine, 26 glycyl-tyrosine, 31 histidine, 46 isoleucine, 104 leucine, 83 lysine, 19 methionine, 40 phenylalanine, 83 proline, 56 serine, 5 taurine, 53 threonine, 21 tryptophan, 8 tyrosine, and 53 valine. Diets were first mixed and then filtered into sterile bags. Just before diet infusion, vitamins (Multi-12K1 Pediatric; Sabex, Boucherville, QC, Canada), minerals (supplied at 200% of the NRC requirement), and lipid (20% Intralipid; Pharmacia, Mississauga, ON, Canada) were added to complete the solution.
Immediately after surgery, the complete diet was infused intravenously via the femoral catheter at 50% of targeted intake until the next morning (day 1). Diet infusion continued via the gastric catheter at 50% of full rate for 12 h, 75% of full rate for another 12 h, and then at full rate (13.5 ml·kg1·h1) until the morning of day 3. On the morning of day 3, piglets were randomly assigned to receive a diet containing a generous (1.80 g·kg1·day1, or 128 mg/g amino acids) or deficient (0.20 g·kg1·day1, or 12 mg/g amino acids) quantity of arginine for 5 days (Fig. 1). To ensure that the diets were isonitrogenous, the concentrations of serine, alanine, and glycine were adjusted in the diet formulations.
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Constant tracer infusions. On the morning of day 5, whole body arginine kinetics were determined by a primed [111 kBq (3 µCi)/kg], constant infusion [185 kBq (5 µCi)·kg1·h1] of L-[guanido-14C]arginine (American Radiolabeled Chemicals, St. Louis, MO) via the gastric catheter. These infusion doses were chosen on the basis of a previous experiment (3). Tracer was infused over a 6.5-h period, with 0.8 ml of blood sampled hourly for the first 4 h and every 30 min thereafter.
On the morning of day 6, intraportal (IP) proline kinetics were determined by a primed [370 kBq (10 µCi)/kg], constant infusion [370 kBq (10 µCi)·kg1·h1] of L-[U-14C]proline (American Radiolabeled Chemicals). IP glutamate kinetics were also determined via a simultaneous primed [185 kBq (5 µCi)/kg], constant infusion [370 kBq (10 µCi)·kg1·h1] of L-[3,4-3H]glutamate (American Radiolabeled Chemicals). Tracers were infused via the umbilical catheter over a 7-h period, with blood sampled every hour for 4 h and every 30 min thereafter. The 1 ml/h infusion of 0.9% saline was discontinued during tracer infusion.
On the morning of day 7, IG proline and glutamate kinetics were determined by a primed [740814 kBq (2022 µCi)/kg], constant infusion [370 kBq (10 µCi)·kg1·h1] of L-[U-14C]proline as well as a simultaneous primed [370 kBq (10 µCi)/kg], constant infusion [370 kBq (10 µCi)·kg1·h1] of L-[3,4-3H]glutamate, respectively. Tracers were infused via the gastric catheter over an 8-h period, with blood sampled hourly for 4 h and every 30 min thereafter; a longer sampling period was used during IG infusions to account for potentially slower appearance of isotope into blood due to gastric emptying and absorption processes. Priming doses for proline and glutamate (21) were determined from previous bolus infusion experiments (unpublished observations).
In five animals, the order of isotope infusion on days 5 and 6 was reversed. These five animals were given the IP primed, constant infusions of L-[U-14C]proline and L-[3,4-3H]glutamate on day 5, and the primed constant infusion of L-[guanido-14C]arginine on day 6 of the protocol. This reversal reduced day 7 background measurements from the IP-infused proline and glutamate tracers by allowing additional washout time.
Analytical procedures. Plasma amino acid concentrations and specific radioactivities (SRA) of glutamate, proline, arginine, hydroxyproline, glutamine, citrulline, ornithine, and urea were measured by reverse-phase HPLC using phenylisothiocyanate derivatives, as previously described (18). Two internal standards, norleucine and L-[4,5-3H]leucine (Amersham Pharmacia Biotech, St. Louis, MO), were added to each 300-µl plasma sample. Postcolumn radioactive derivatives were collected in 2-ml fractions to which 14 ml of scintillant were added. Radioactive fractions were counted on a liquid scintillation counter using the dual-isotope counting method for 3H and 14C. On days 6 and 7 of the infusions, SRA values at plateau were corrected for background SRA values measured immediately before the respective infusions.
Plasma ammonia concentrations were determined every 24 h during test diet infusions. The colorimetric assay used in the analysis was based on the amination of 2-oxoglutarate to glutamate with simultaneous oxidation of NADPH (Sigma Procedure No. 171-UV, Sigma Diagnostics, St. Louis, MO). Plasma urea concentrations were measured daily and during tracer infusions by use of a spectrophotometric assay kit in which ammonia was liberated from urea by enzymatic hydrolysis (Sigma Chemical).
Plasma NOx concentrations were determined every 24 h during test diets via chemical reduction of nitrate to nitrite, followed by spectrophotometric analysis of total nitrite with Griess reagent (Oxis International, Portland, OR).
Calculations.
Plasma SRAs were calculated as plasma SRA (Bq/mmol) = amino acid radioactivity (Bq/l)/amino acid concentration (mmol/l). Plateau in plasma SRA was calculated as the mean of the SRA for the time points in the plateau period. All plateaus included at least four time points except for the conversion of arginine to urea, which included three. At plateau, SRA ratios of product to precursor were used to determine the fractional net conversion of precursor to product:
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Fluxes of infused amino acids (arginine, proline, and glutamate) were calculated as:
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Values for flux and fractional net conversion were used in the calculation of molar rates of conversion of precursor (proline, glutamate) to arginine, following the approach of Beaumier et al. (2):
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Small intestinal (gut) first-pass contribution to Qprecursor to product was calculated as the difference between values for Qprecursor to product during IG and IP isotope infusions.
Statistical analyses. The dependent variables NO, ammonia, and BUN were analyzed using the Mixed-model procedure of SAS version 8.3 (SAS Institute, Cary, NC) with treatment (generous or deficient arginine diet) as a fixed effect, days as a fixed repeated effect, and pig (treatment) as a random error term to test treatment. The variance-covariance matrix was chosen for the statistical model using an iterative process wherein the best fit was based on Schwarz's Bayesian Criterion. The Kenward-Roger option was used to determine denominator degrees of freedom. Least square means were separated using the pdiff option for significant (P < 0.05) fixed effects and are presented throughout the paper. A one-tailed test was used to compare plasma ammonia and BUN values between diet groups, because we expected these values to be significantly higher in arginine-deficient animals compared with those receiving a generous quantity of dietary arginine. Given the lack of comparable studies, a prediction could not be made regarding the results for the nitric oxide (NO) analysis; hence, a two-tailed test was employed.
The dependent variables of plasma net flux from proline to arginine (QProArg) and the plasma fractional net conversion of proline to hydroxyproline, citrulline, glutamine, and ornithine were analyzed using a two-by-two factorial design with interaction using the Mixed model procedure of SAS (SAS version 8.3). The fixed effects were treatment (generous or deficient arginine diet) and route of isotope infusion (IP or IG). A two-way analysis of variance with interaction was used to test whether the SRA values calculated during the tracer infusions on day 7 were significantly affected by the order of isotope infusion on days 5 and 6 (SAS version 8.3). All other comparisons between diet groups were performed using a two-tailed t-test (SAS version 8.3). Data are presented as means with pooled SE and were considered significantly different if P < 0.05.
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RESULTS |
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The recovery of radioactive label from glutamate in arginine was not different from zero when corrected for background. The net flux from glutamate to arginine (QGluArg) was therefore deemed to be biologically insignificant (<1% of arginine flux). As a result, fractional net conversions of glutamate to hydroxyproline, glutamine, citrulline, and ornithine were not calculated.
Plasma NOx concentrations were not affected by the level of arginine provided in the diet (P < 0.09; Fig. 4).
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DISCUSSION |
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Despite twofold greater whole body arginine synthesis in deficient compared with arginine-supplemented animals (Table 4), the total available arginine (intake, 0.20 g·kg1·day1 + synthesis, 0.68 g·kg1·day1) in the deficient animals was not sufficient to satisfy the metabolic requirement, as evidenced by elevated plasma ammonia (Fig. 2) and BUN levels (Fig. 3). Therefore, intestinal arginine synthesis was not sufficiently upregulated to compensate for the dietary arginine deficiency and must represent the maximum arginine synthetic capacity of the neonatal piglet intestine in this model. Plasma proline concentrations indicate that arginine synthesis was not limited by supply of dietary proline but by limited de novo synthetic capabilities. In conditions that increase the metabolic demand for arginine beyond this maximum arginine synthetic capacity, dietary arginine supplementation is necessary.
Our second objective was to quantify whether there is a basal level of arginine synthesis that occurs even when there is an adequate supply of arginine. This objective was achieved by providing a generous quantity of arginine in the diet (8, 30). Theoretically, arginine synthesis should be minimized under such conditions. These piglets, however, synthesized 0.36 g·kg1·day1 arginine, about one-half as much as deficient pigs.
Recent results from our laboratory (3, 8, 30) have demonstrated that indexes of arginine adequacy reach a plateau when arginine is provided between 1.20 and 2.00 g·kg1·day1, suggesting that the total metabolic demand for arginine is satisfied at 1.20 g·kg1·day1, a level three times greater than the current NRC-estimated dietary requirement of 0.38 g·kg1·day1 (22) and almost double the arginine intake from sow's milk (0.66 g·kg1·day1) (13). On the basis of these previous observations, we are reasonably certain that 1.80 g·kg1·day1 arginine is metabolically sufficient and likely a generous intake for the neonatal piglet. We have therefore demonstrated that there is a basal and obligatory rate of arginine synthesis in the neonatal piglet.
The dietary regulation and partitioning of precursors for arginine synthesis formed the third objective in this study. Although renal arginine synthesis does not appear to be under dietary regulation in the adult human (9, 10) or in the rat (14), our results suggest that whole body QProArg in the neonate is under dietary regulation. When challenged by an inadequate supply of dietary arginine, piglets were able to upregulate net synthesis of arginine from proline. On a whole body level, the net quantity of arginine synthesis from proline doubled in deficient compared with adequately fed piglets and tripled during IP infusions (Table 4). The quantity of arginine synthesized by the gut on first pass, however, was not different between diet levels. These data suggest that first-pass gut arginine synthesis occurred at its maximum capacity regardless of the dietary inclusion level of arginine. Because first-pass gut conversion of proline to arginine was already operating at its maximum capacity, piglets provided with a deficient quantity of arginine in the diet upregulated conversion of proline to arginine by some other mechanism to match the level of conversion occurring on first pass by the gut.
The IP infusion of nutrients was developed in our laboratory to isolate first-pass gut metabolism (4). We have previously used this method to demonstrate that conversion of proline to arginine is dependent on first-pass gut metabolism in the neonate (3); however, we did not previously investigate the contribution of the liver or of other tissues to arginine synthesis. Increased conversion of proline to arginine during IP infusion between test diets may result from increased arterial extraction of proline by the healthy gut for arginine synthesis. This arterial extraction of proline may explain why, in an earlier study, piglets fed IG an arginine- and/or proline-free diet exhibited a less severe rise in plasma ammonia than piglets fed the same diet intravenously (7). Because our data suggest that dietary glutamate is not an important precursor for arginine synthesis in piglets of this age, it is possible that the healthy gut of the IG-fed animals was extracting arterial proline for conversion to arginine.
Although these data suggest that the liver and the kidney are not involved in neonatal arginine synthesis under normal conditions, urea cycle enzyme and cationic amino acid transporter activities may be altered under abnormal dietary conditions. There is evidence that factors such as starvation, high-protein feeding, and glucagon treatment can increase the hepatic activities and mRNA levels of all urea cycle enzymes (19). In the rat kidney, high-protein feeding causes a doubling in the activity of "arginine synthetase" [argininosuccinate synthase (ASS) + argininosuccinate lyase (ASL)] (27) and in the relative abundance of mRNA for the enzymes of arginine synthesis (20). Furthermore, a number of urea cycle enzymes have extrarenal and extrahepatic locations (25, 31). Windmueller and Spaeth (31) observed net citrulline release from the skeletal muscle in the rat and also found evidence of extrarenal and extrahepatic citrulline utilization. Whether or not these other tissues can make a significant contribution to arginine synthesis is not known. However, it must be noted that, although the kidney is the major site of arginine synthesis in the adult and renal arginase activity is low relative to the liver (15), the activity of ASS and ASL is low in the kidney of the neonate (35).
The recovery of 3H in arginine from [3H]glutamate was not detectable by our methods; our detection limits correspond to a conversion of no more than 1% of arginine flux. Our conclusions regarding the effectiveness of glutamate as a precursor for arginine concur with findings by Murphy et al. (21) in neonatal piglets and by Wu et al. (36) and Blachier et al. (5) in neonatal piglet enterocytes. Furthermore, piglets fed a deficient level of dietary arginine had 0.88 g·kg1·day1 of available arginine from a combination of diet and intestinal synthesis from proline; however, these animals still experienced episodes of hyperammonemia, a phenomenon that would not have occurred if they were synthesizing significant quantities of arginine from glutamate. Because of the marked changes in the activities of the enzymes that synthesize arginine from glutamate and glutamine during the course of early development (33), our conclusions regarding the suitability of glutamate as a precursor for arginine synthesis are age specific and may apply only to a young piglet.
IG arginine fluxes were used in this study for calculations of intestinal arginine synthesis. Because arginine is synthesized in the gut of neonates, it is not reasonable to exclude the effects of the gut from our conversion estimates. IG flux represents whole body flux, and oral dosing includes metabolism by the gut, which contributes to the central plasma pool. In particular, this inclusion is necessary because the "extraction" of amino acids by the gut is not irreversible, as is often assumed for a short infusion period. In fact, van der Schoor et al. (28) have recently showed that almost 50% of the lysine in the portal vein is derived from recycling of glycoproteins by the gut within a 6-h period; so orally delivered isotope is "extracted" by the gut, incorporated into proteins to be secreted into the lumen, and then degraded such that the extracted amino acids eventually enter the systemic circulation and central plasma pool. Therefore, subtracting this extraction from the infusion dose is not appropriate, given that this isotope is eventually reintroduced into the plasma pool. Regardless of whether intravenous, IP, or IG fluxes are used for calculation purposes, the relative differences between diets in QProArg are real. Indeed, if splanchnic extraction is subtracted from the IG infusion dose to determine intravenous flux rates in the pigs of the present study (2), then the data would be approximately one-half in absolute value but would not change the statistical comparisons made. The major findings of this research confirm the existence of a basal and a maximal rate of arginine synthesis and of dietary regulation of arginine synthesis from proline. These findings are independent of specific flux values.
In this study, there was a significant difference in the rate of body weight gain in animals infused IG between diets containing generous or deficient levels of arginine. This difference was not, however, in the expected direction, because the piglets fed a deficient diet gained significantly more body weight than those fed an adequate arginine diet. The higher plasma ammonia and BUN levels in these deficient piglets, however, suggest more net catabolism of protein in this group. The data of House et al. (17) are instructive in this respect. In that study, glutamine supplementation to total parenteral nutrition tended to increase live body weight gain in neonatal piglets via an increase in total body water and, in particular, extracellular fluid expansion. The formation of glutamine from glutamate and ammonia is an important physiological means of scavenging excess ammonia. Because the urea cycle function of the arginine-deficient diet group was challenged by an inadequate arginine intake, the animals exhibited plasma glutamine concentrations that were fourfold greater than those of piglets fed an adequate level of arginine. Therefore, high plasma glutamine concentrations in deficient animals may have induced an expansion in the extracellular fluid, resulting in greater body weight gain.
Animals fed a deficient diet had plasma arginine concentrations that were 90% lower than those observed in the animals fed a generous quantity of the amino acid, similar to the pattern of the decline in the dietary arginine concentration. Despite plasma arginine concentrations that were much lower than those in the sow-fed reference group, plasma ornithine concentrations of the arginine-deficient piglets did not drop below levels characteristic of sow-fed piglets, although they were significantly lower than those of adequately fed animals. These observations are similar to those made by Castillo et al. (11), who observed a decline in ornithine oxidation in healthy men fed arginine-free diets. A reduction in ornithine oxidation could help to preserve urea cycle substrates in the absence of sufficient dietary arginine. Plasma citrulline concentrations were unaffected by dietary arginine deficiency, a phenomenon that has also been documented in rats (16), 20- to 50-kg pigs (24), and adult humans (12).
There may have been a trend suggestive of impaired NO synthesis in arginine-deficient animals in the present study. In rats, Wu et al. (34) observed a reduction in NO synthesis by constitutive NO synthase and inducible NO synthase during arginine deficiency, although the magnitude of the response was smaller in the rats than in the present study. To the authors' knowledge, no comparable studies of the influence of dietary arginine on NO synthesis exist in healthy neonates. That dietary arginine deficiency may have an impact on NO production is of extreme importance to the health of neonates, because decreased NO production is associated with neonatal diseases, such as persistent pulmonary hypertension of the newborn and necrotizing enterocolitis (NEC). Indeed, plasma arginine concentrations are lower in infants affected with these conditions (1, 23, 29, 39), and arginine supplementation can significantly reduce the incidence of NEC (1). Conversely, overproduction of NO can contribute to tissue injury, hypotension, and myocardial failure (6).
In conclusion, although first-pass gut metabolism is responsible for 4263% of whole body arginine synthesis, the gut is incapable of upregulating proline-to-arginine conversion (on first pass) during arginine deficiency. Therefore, the role of first-pass gut metabolism in the synthesis of arginine, although substantial, seems to be limited to an obligatory function. This lack of dietary response compares with the more than threefold increase in proline-to-arginine conversion when first-pass gut metabolism is bypassed (i.e., IP). These data suggest that upregulation of proline-to-arginine conversion occurs via increased arterial extraction of proline by the gut or in nonintestinal tissues. This study has demonstrated that dietary arginine is an important regulator of endogenous arginine synthesis in the neonatal piglet. In addition, because glutamate was not a significant precursor for arginine synthesis, proline is the primary precursor for neonatal arginine synthesis, further supporting the coindispensability (7) of these amino acids in the neonate.
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GRANTS |
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ACKNOWLEDGMENTS |
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Current address for R. F. P. Bertolo and J. A. Brunton: Department of Biochemistry, Memorial University of Newfoundland, St. John's, NL, Canada A1B 3X9.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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