Acid Uptake by the Human - NCBI - NIH

ANNALS Of: SURGERY V'ol. 2 19. No. 6. 7 1 5-724 c 1994 J. B. Lippincott Companm

Growth Hormone Enhances Amino Acid Uptake by the Human Small Intestine Yoshifumi Inoue, M.D.,* Edward M. Copeland, M.D., F.A.C.S.,* and Wiley W. Souba, M.D., ScD., F.A.C.S.t From the Department of Surgery, University of Florida College of Medicine,* Division of Surgical Oncology, Gainesville, Florida, and the Department of Surgery, Massachusetts General Hospital and Harvard Medical School, t Boston, Massachusetts

Objective The effects of growth hormone (GH) on the luminal transport of amino acids and glucose by the human small intestine were investigated.

Summary Background Data The anabolic effect of growth hormone administration is associated with nitrogen retention and an increase muscle strength, but the impact of growth hormone on nutrient uptake from the gut lumen has not been examined.

Methods Twelve healthy patients received a daily subcutaneous dose of low-dose GH (0.1 mg/kg), highdose GH (0.2 mg/kg), or no treatment (controls) for 3 days before surgery. At operation, ileum (8 patients) or jejunum (4 patients) was resected, and brush border membrane vesicles (BBMVs) were prepared by differential centrifugation. Vesicle purity was confirmed by a 16-fold enrichment of marker enzymes. The carrier-mediated transport of glutamine (System B), leucine (System L), alanine (System B), arginine (System y+), MeAIB (methyl a-aminoisobutyric acid [System A]), and glucose (Na+-dependent glucose transporter) by BBMVs was measured by a rapid mixing/ filtration technique.

Results Treatment with low-dose GH resulted in a statistically insignificant increase in amino acid transport rates in jejunal and ileal BBMVs. High-dose GH resulted in a generalized 20%- to 70%- stimulation of amino acid transport, whereas glucose transport was not affected. The effects of GH were similar in ileum and jejunum. Kinetic analysis of the transport of glutamine (the most abundant amino acid in the body and the principal gut fuel) and the essential amino acid leucine revealed that the increase in transport was caused by a 50% increase in carrier Vmax, consistent with an increase in the number of functional carriers in the brush border membrane. Pooled analysis of transport velocities demonstrated that total rates of amino acid uptake from the gut lumen were increased significantly by 35% in GH-treated patients.

Conclusions The ability of GH to enhance amino acid uptake from the gut lumen provides energy and precursors for protein synthesis in the gut mucosa, as well as additional substrate for anabolism in other organs.

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The mucosa of the small intestine plays an essential role in the absorption of luminal nutrients. Uptake of amino acids and glucose across the brush border is accomplished by distinct transporter proteins, the majority of which require sodium cotransport.' Nutrient uptake supplies amino acids for synthetic pathways in the gut mucosa and provides substrate for peripheral tissues. Failure to consume adequate amounts of dietary amino acids, either because of inadequate intake or because of an "injured" gut mucosa, leads to erosion of lean body mass and negative nitrogen balance. In the long run, the patient can become so malnourished that wound healing is impaired, susceptibility to infection is increased, and muscle strength is diminished. Although the initial enthusiasm for providing specialized nutrition to critically ill surgical patients was great, several reports in the past decade indicate that aggressive nutritional support does not prevent negative balance and erosion of lean body mass during severe catabolic illness.2 Although it is clear that enteral nutrition is superior to parenteral nutrition, any strategy of enhancing amino acid uptake by the intestine could benefit the catabolic surgical patient. One anabolic agent that currently is available for clinical use is recombinant human growth hormone (GH), a single chain polypeptide of 191 amino acids. It is the most abundant hormone in the pituitary gland, and its growth-promoting properties are well known. The anabolic effect of GH administration is associated with nitrogen retention and is reflected by a diminished urea excretion that results from a true reduction in urea production in the liver.3 In addition, administration of GH to catabolic surgical patients or to malnourished patients who require nutritional rehabilitation has been shown to accelerate protein gain and muscle strength.46 The effects of GH on nutrient uptake from the gut lumen have not been examined. In this investigation, we examined the effects of GH administration on small intestinal brush border amino acid and glucose transport activity in humans. We hypothesized that increases in the intrinsic activity of brush border carrier proteins underlie a potential mechanism at the level ofthe plasma membrane, whereby mucosal nutrient uptake is enhanced by GH administration.

MATERIALS/METHODS Reagents and Chemicals All chemicals and reagents used were of analytical quality and were purchased from Sigma Chemical ComSupported by NIH Grant CA 45327 and a grant from the GI Study Section ofthe Veterans Administration Merit Review Board (Dr. Souba). Presented at the 105th Meeting of the Southern Surgical Association, The Homestead, Hot Springs, Virginia, December 5-8, 1993. Address reprint requests to Wiley W. Souba, M.D., Sc.D., Chief, Division of Surgical Oncology, Massachusetts General Hospital, Cox Building, 100 Blossom Street, Boston, MA 02114. Accepted for publication January 4, 1994.

Ann. Surg. *-June 1994

pany (St. Louis, MO). Radiolabeled L-glutamine, L-alanine, L-leucine, L-arginine, L-MeAIB (methyl a-aminoisobutyric acid) and D-glucose were purchased from Amersham (Arlington Heights, IL). Human methionyl recombinant GH was provided as a gift from Genentech, Inc. (San Francisco CA).

Patient Selection Adult surgical patients admitted to the Veterans Administration Hospital in Gainesville, Florida, were eligible to participate in the study. The studies were approved by the Institutional Review Board at the University of Florida College of Medicine and by the Subcommittee for Clinical Investigation at the Gainesville Veterans Administration Medical Center. Segments of small intestine (- 10 cm) were obtained intraoperatively from 12 healthy patients. Patients were randomized to receive preoperatively low-dose GH (0.1 mg/kg daily subQ X 3 days), high-dose GH (0.2 mg/kg/ day X 3 days), or no treatment (controls). Jejunum was obtained from four patients (two controls, one low-dose GH, one high-dose GH) and ileum was obtained form eight patients (three controls, one low-dose GH, four high-dose GH). Patients receiving GH from whom jejunum was harvested underwent Roux-en-Y diversion. Control jejunum was obtained from patients in whom normal jejunum was resected en bloc with other tissues. Ileum was obtained from patients undergoing right hemicolectomy for cecal or ascending colon lesions. None of the patients had weight loss or clinically significant organ dysfunction. All patients were consuming a regular diet and received nothing by mouth for 24 hours before operation. The small intestine was not devascularized until immediately before it was passed off the operating table, at which time it was placed promptly on ice and transported immediately to the research laboratories. The mucosa was rinsed with 0.9% ice-cold saline, scraped with a glass slide, and stored in liquid nitrogen. Brush border membrane vesicles (BBMVs) from human small intestinal mucosa were prepared as described in the following section.

Membrane Vesicle Preparation Brush border membrane vesicles were prepared by an Mg++ aggregation/differential centrifugation tech-

nique.7'8 The previously frozen mucosa first was thawed, and all subsequent steps of the preparation were conducted at 0 C to 5 C. Briefly, each gram of mucosal scrapings was homogenized in buffer, and the supernatant containing brush border material was collected and centrifuged. The final pellet was resuspended in the same buffer to yield a final protein concentration of 10 mg/ mL to 15 mg/mL. The brush border enzymes alkaline

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phosphatase and F-glutamyl transpeptidase were measured routinely to assay for brush border vesicle purity.9 Protein concentration was determined by the Biorad protein assay (Bio-Rad Laboratories, Richmond, CA), with gamma globulin as the protein standard.

Growth Hormone Enhances Intestinal Amino Acid Transport

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and F-glutamyl transpeptidase.7 In the present study, both enzyme markers demonstrated a statistically significant 15- to 17-fold enrichment in vesicles, compared with the crude mucosal homogenate.

Transport Characteristics Transport Measurements The uptake of 3[H]-labeled substrate was measured using a rapid mixing/filtration technique.7 The transport of glutamine (System B), alanine (System B), leucine (System L), arginine (System y+), the amino acid analog MeAIB (System A), and glucose (Na+-dependent glucose transporter) was assayed. For each uptake measurement, 10 ,AL of BBMVs and 40 ,uL of the radioactive uptake buffer were placed separately at the bottom of a 12 X 75 polystyrene tube (Fisher Scientific Inc., Pittsburgh, PA). The uptake buffer components were adjusted so that the final concentration mixture contained initial gradients of 120 mM NaCl or KCI and labeled substrate at 10- to 50-,uM concentrations. The reaction was initiated by rapidly vibrating the tube, and, after the prescribed reaction period (15 seconds-I hour), 1 mL of ice-cold stop buffer was added to quench the reaction. The quenched reaction mixture then was filtered, using a prewetted and chilled O.45-,um membrane filter. The membranes were washed once with 5 mL of stop buffer and then dissolved in Aquasol Scintillation cocktail (Dupont, New England Nuclear Research Products, Boston, MA). The radioactivity trapped by the vesicles was measured by liquid scintillation counting. Values for nonspecific retention of the radioactivity by the filter and the vesicles were obtained from time-zero uptakes and were subtracted from the total filter radioactivity. The radioactivity was converted to units of uptake and expressed as picomoles per milligram protein per time. Uptake was measured in the presence and absence of sodium with potassium as the control cation.

Statistical Analysis All data are expressed as mean ± standard error. Data compared using analysis of variance with the appropriate post-hoc test where indicated. (MacIntosh LCII Computer, Statview 512 Statistical Program, Apple Computers, Inc.). A p value of less than 0.05 was considered statistically significant.

were

RESULTS Brush Border Purification The purity of our brush border membranes has been ascertained previously by determining the activities of the brush border marker enzymes alkaline phosphatase

Transport of glutamine and alanine, MeAIB, and glucose were mediated by the Na+-dependent transport systems B, A, and the Na+-dependent glucose carrier, respectively. Arginine and leucine transport were mediated by the Na+-independent Systems y+ and L, respectively. The Na+-dependent transport of glutamine and alanine exhibited classical overshoots. Vesicular size was similar in all compared groups, as evidenced by identical 1-hour equilibrium values (Fig. 1). Osmolarity studies (data not shown) showed uptake into an osmotically active intravesicular space.

Effects of Growth Hormone on Transport Activity The 10-second jejunal transport of substrates by hepatic plasma membrane vesicles (HPMVs) from control and GH-treated patients is shown in Figure 2. Low-dose treatment did not alter transport, whereas high-dose treatment appeared to result in a generalized increase in jejunal amino acid transport activity, with no effect on glucose uptake. The effect was greatest for the amino acids glutamine, arginine, and leucine. Similar results were observed in ileal BBMVs (Fig. 3). Low-dose treatment with GH did not alter transport activity, but highdose treatment resulted in a statistically significant increase in the transport of glutamine (p < 0.05 vs. control), leucine (p < 0.05 vs. control), and alanine (p < 0.05 vs. control). There was a trend toward an increase in System y+ (arginine) and System A (MeAIB) activity but this did not reach statistical significance. Glucose transport activity was not altered. Pooled transport data from ileal BBMVs studies are shown in Figure 4. To investigate the nature of the increase in the ileal transport of glutamine by BBMVs from patients treated with high-dose GH, kinetic studies were undertaken (Fig. 5). The Na+-dependent initial rate of glutamine transport by vesicles from control and high-dose GHtreated patients was determined across increasing extravesicular glutamine concentrations from 50 AuM to 5 mM. Representative data are shown in Figure 5A. The initial glutamine transport rate by vesicles from patients treated with high-dose GH was increased across all concentrations relative to that of vesicles from control patients. Eadie-Hofstee linear transformation of the data (Figure SB) demonstrated that the increase in transport activity was secondary to a 52% increase in the maximal velocity of transport, with no alteration in transporter

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affinity. Similarly, the carrier-mediated Na+-independent initial rate ofleucine transport by vesicles from control and high-dose GH-treated patients was determined across increasing extravesicular concentrations from 50 ,uM to 10 mM (Fig. 6). Representative data are shown in Figure 6A. The initial leucine transport rate by vesicles from patients treated with high-dose GH was increased across all concentrations relative to that of vesicles from control patients. Linear transformation of the data (Figure 6B) demonstrated that the increase in transport activity was secondary to a 45% increase in the maximal velocity of transport, with no alteration in transporter

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Glutamine Arginine Leucine MeAIB Alanine Glucose Figure 2. Ten-second jejunal amino acid and glucose transport rates expressed as a percentage of transport in control BBMVs. Brush border membrane vesicles were incubated in 100 AM each of the indicated substrates and transport assayed. The bar graph is the mean of two assays performed separately in triplicate on BBMVs preparations from two control, one low-dose GH-treated patient, and one high-dose GH-treated patient.

Time (min)

affinity. Similar studies were done using MeAIB (Fig. 7). Kinetic parameters for MeAIB were not altered significantly. Pooled results from all amino acid transport studies are shown in Figure 8. Low-dose GH treatment caused a slight (8%) increase in transport activity (p = NS), whereas administration of the higher dose increased total amino acid transport rates by about 35% (p < 0.05 vs. controls).

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Figure 1. Time course of Na+-dependent ileal glutamine (A), leucine (B), and MeAIB (C) uptake by BBMVs from control and GH-treated (0.2 mg/kg X 3 days) patients. Brush border membrane vesicles were incubated with uptake buffer and transport assayed as described in Methods. Each data point represents mean ± SEM of triplicate measurements. When not shown, the error bars are contained within the symbol.

Luminal amino acids and glucose are transported across the brush border membrane of the small intestine by several well-described transport systems.' In general, the movement of luminal substrates into the cytoplasm of the enterocyte occurs via three separate pathways-a sodium-dependent route, a sodium-independent pathway, and by diffusion, which reflects the permeability of the membrane. Three principle factors determine the uptake of amino acids and glucose across the brush border. They are the quantity of luminal substrate available to the epithelial cells, the capacity of the individual cells to translocate the compound into the intracellular compartment, and the capacity of the cell to use intracellularly available substrate. In our investigations, we focused on the intrinsic activity of the transporter protein by measuring transport rates in BBMVs. The use of plasma membrane vesicles to assess brush border substrate transport activity offers several advantages from other approaches.' Alterations in membrane transport activity are preserved during the preparation of vesicles,


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Figure 3. Ten-second ileal amino acid and glucose transport rates in jejunum form three control patients, one patient receiving low-dose (0.1 mg/kg X 3 days) GH, and four patients receiving high-dose (0.2 mg/kg X 3 days) GH. Brush border membrane vesicles were incubated in 100 ,M each of the indicated substrates and transport assayed. The bar graph is the mean of three assays performed separately in triplicate on BBMVs preparations. *p < 0.05 vs. control.

and transport activity can be evaluated apart from other confounding influences, such as metabolism and transstimulation/inhibition. Our BBMVs demonstrated enrichments and overshoots, indicating vesicle purity and usefulness. Because surgical patients develop erosion of lean body mass, which can be associated with intestinal dysfunction, efforts have focused on methods of modifying the

stress response in the hopes of improving clinical outcome. '° Despite attempts to demonstrate otherwise, sev-

eral reports indicate that aggressive nutritional support does not prevent considerable loss of lean body mass during critical illness.2 Consequently, attempts have focused on methods of attenuating the catabolic response employing a combination of hormonal supplementation with nutritional intervention.4 6 "1Many of these investigations have focused on the anabolic agent GH, in large part because of advances in molecular biology, which have made available the technology for the production y Glucose of relatively large quantities of the compound. We studied the effects of GH on amino acid transport by the huH Leucine o 1man small intestine. We measured amino acid and glu* * cose transport rates in BBMVs prepared from surgical patients receiving GH before surgery. The amino acids 3 Alanine we chose to study are transported by distinctly different 0 transport proteins, System B (glutamine and alanine), System L (leucine), System y+ (arginine), and System A (MeAIB); this allowed us to compare the effects of GH on a variety of carriers. Control 14 TI The classical anabolic properties of GH have been well 100 200 established. They include nitrogen retention associated 150 50 0 with an increase in protein synthesis and fat mobilizaRelative Uptake (% of Control) tion. The nitrogen retention is reflected by a diminished Figure 4. Pooled ileal transport rates in BBMVs from control and highurea excretion that results from a true reduction in hedose GH-treated patients. There was a generalized increase in amino acid urea production.3 More recent studies examining patic transport activity in patients receiving GH, whereas glucose transport was the influence ofhuman recombinant GH administration unaffected. Each data point represents the mean SEM of triplicate meain catabolic patients have demonstrated marked imsurements. *p < 0.05 vs. control.

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transport as a regulator of amino acid metabolism, we focused our attention on the brush border membrane in the present investigation. Global increases in amino acid transport activity were observed in GH-treated patients. When the genes that encode these specific proteins are cloned, and antibodies to these carriers are purified, the molecular regulation of intestinal transport can be studied. The mechanism by which GH increase amino acid transport activity also is unclear, but may be related to the effects of GH on insulin-like growth factor- 1 produc-

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ing.4'6"2 Much attention regarding the mechanisms that underlie this improvement in nitrogen retention has focused on the effects ofGH in stimulating muscle protein synthesis. Considerably less is known about the role of the gut in amino acid metabolism during GH therapy. The role of membrane transport in the regulation of amino acid metabolism has become increasingly apparent in recent years. In light of the gross observations on nitrogen retention elicited by GH therapy and the accumulating evidence supporting the role of membrane

Velocity/lLeucinel Figure 6. Kinetic analysis of carrier-mediated leucine transport activity by BBMVs from control and GH-treated patients. Patients received 0.2 mg/kg (high-dose) GH or no treatment for 3 days preoperatively, and membrane vesicles were prepared from ileal mucosa harvested at surgery (A). The Na+-independent 10-second transport of [3H]-leucine was determined as a function of increasing extravesicular leucine concentrations. (B) EadieHofstee linear transformation of the data was performed by plotting transport velocity as a function of velocity/leucine concentration. Regression analysis of the resultant plots revealed maximal transport velocity (V,., = y-intercept) and transporter affinity (Km = negative slope). Kinetic parameters were: Vms,,, 155 pmol/mg protein/i 0 sec in control vs. 215 in GH; and Km, 424 ,M in control vs. 416 in GH. Representative plots are shown.


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Because glutamine is the principal small intestinal fuel and the most abundant amino acid in the body,'4 we examined the effect of GH on glutamine transport kinetics. In addition, we studied transport kinetics for the essential amino acid leucine. Growth hormone did not change the affinity of the Na+-dependent cotransporter for glutamine or leucine (the apparent Km was unchanged) but the VNax increased by 50%. This is consistent with a GHstimulated increase in the number of functional carriers in the brush border membrane. In addition, the transport of alanine was accelerated, and there was a trend toward an increase in arginine transport. One question raised by these studies is whether lumi-

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nal transport activity can be further augmented with a combination of GH and high nitrogen feeding. The activity of brush border nutrient transporters can be regulated by their respective substrates.'5"6 Diamond and Karasov'7 studied the influence of diet on the adaptive regulation of intestinal nutrient transporters. Their work indicated that the basal rate of luminal transport can be unregulated by dietary substrate. This observation can be important in critically ill patients because it suggests that enteral nutrition can help offset the fall in luminal transport activity that occurs during severe infection.7 Maintenance of transport function also can be important in such individuals because dietary substrate can profoundly influence protein synthesis in the gut mucosa. Furthermore, enteral feedings improve outcome in critically ill patients. As recent work indicates that mucosal protein synthesis is increased after induction of fecal peritonitis by cecal ligation and puncture,'8 GH administration may augment intestinal amino acid uptake in critically ill patients to support mucosal synthetic requirements. Further studies will be necessary to define the combined benefits of GH and aggressive enteral feedings. Together with previous studies that demonstrate the GH diminishes hepatic amino acid transport'9 and ureagenesis,3 the current study indicates that GH redistributes the flow of amino acids in a way that can support anabolism in peripheral tissues (Fig. 9). Growth hormone accelerates luminal amino acid transport rates while reducing transport activity in the liver. The net result is an increased availability ofamino acids to support protein synthesis in peripheral tissues, such as skeletal muscle. Regardless of how GH works, these studies demonstrate that the small intestine is a not simply a passive participant in the overall improvement in nitrogen balance which accompanies GH administration, but is an

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active organ that can participate in the redistribution of amino acid nitrogen to peripheral tissues.

10. Wilmore DW. Catabolic illness: strategies for enhancing recovery. New Engl J Med 1992; 325:695-702. 11. Wilmore DW, Moylan JA, Bristow BF, et al. Anabolic effects of human growth hormone and high caloric feedings following thermal injury. Surg Gynecol Obstet 1974; 138:875-883. 12. Herndon DN, Barrow RE, Kunkel KR, et al. Effects of recombinant growth hormone on donor-site healing in severely burned children. Ann Surg 1990; 212:424-43 1. 13. Daughaday WH, Rotwein P. Insulin-like growth factors I and II: peptide, messenger ribonucleic acid and gene structures, serum and tissue concentrations. Endocr Rev 1989; 10:68-91. 14. Windmueller HG. Glutamine utilization by the small intestine. Adv Enzymol 1982; 53:202-231. 15. Ferraris RP, Diamond JM. Specific regulation of intestinal nutrient transporters by their dietary substrates. Annu Rev Physiol 1989; 51:125-139. 16. Salloum RM, Souba WW, Fernandez A, et al: Dietary modulation of small intestinal glutamine transport in intestinal brush border membrane vesicles of rats. J Surg Res 1990; 48:635-638. 17. Diamond JM, Karasov WH. Adaptive regulation of intestinal nutrient transporters. Proc Natl Acad Sci U S A 1987; 84:2242-2245. 18. Von Allmen D, Hasselgren PO, Higashiguchi T, et al. Increased intestinal protein synthesis during sepsis. Surg Forum 1990; 41: 68-69. 19. Pacitti AJ, Inoue Y, Plumley DA, et al. Growth hormone regulates amino acid transport inn human and rat liver. Ann Surg 1992; 216: 353-362.

Discussion

References 1. Souba WW, Pacitti AJ. How amino acids get into cells: mechanisms, models, menus, and mediators. J Parent Ent Nutr 1992; 16: 569-578. 2. Streat SJ, Beddoe AH, Hill GL. Aggressive nutritional support does not prevent protein loss despite fat gain in septic intensive care patients. J Trauma 1987; 27:262-266. 3. Welbourne T, Joshi S, McVie R. Growth hormone effects on hepatic glutamate handling in vivo. Am J Physiol 1989; 257:E959E962. 4. Byrne TA, Morrissey TB, Gatzen C, et al. Anabolic therapy with growth hormone accelerates protein gain in surgical patients requiring nutritional rehabilitation. Ann Surg 1993; 218:400-418. 5. Jiang ZM, He GZ, Zhang SY, et al. Low-dose growth hormone and hypocaloric nutrition attenuate the protein-catabolic response after major operation. Ann Surg 1989; 210:513-525. 6. Ziegler TR, Young LS, Manson JM, Wilmore DW. Metabolic effects of recombinant human growth hormone in patients receiving parenteral nutrition. Ann Surg 1988; 208:6-16. 7. Salloum RM, Copeland EM, Souba WW. Brush border transport of glutamine and other substrates during sepsis and endotoxemia. Ann Surg 1991; 213:401-410. 8. Stevens BR, Kaunitz J, Wright EM. Intestinal transport of amino acids and sugars: advances using membrane vesicles. Ann Rev Physiol 1984; 417:417-433. 9. Mircheff AK, Wright EM. Analytical isolation of plasma membranes of intestinal epithelial cells, identification of Na-K ATPase, and the distribution of enzyme activities. J Membrane Biol 1976; 28:309-315.

DR. BASIL A. PRUITT, JR. (San Antonio, Texas): I rise to compliment Dr. Souba and his colleagues on another meticulously performed and conservatively interpreted study that extends their research program focused on the key area of protein metabolism in various subsets of surgical patients. In this study, they have described the actions of recombinant growth hormone on amino acid transport systems in the enterocyte and illuminated a mechanism which appears to contribute to the overall beneficial effect of growth hormone on nitrogen economy. Several years ago, Dr. Wilmore and others at our laboratory reported that in hypermetabolic burn patients human growth hormone had a nitrogen sparing effect that was dose related and dependent upon carbohydrate intake meeting measured metabolic needs. These data appear to integrate those two findings and emphasize the importance of enteral protein transport in growth hormone related preservation of lean body mass. Answers to several questions may help us evaluate the authors' conclusions. In the liver, there are dual transport systems for glutamine and arginine, one sodium dependent and one sodium independent. In the enterocyte, are there also dual systems, and if so, is the effect of growth hormone of even greater magnitude than that measured in this study? Since the effect of growth hormone appears to be related to nutritional status, were the patients receiving adequate nutrition and did their actual weights compare favorably with ideal weight? Since growth hormone therapy has been associated with hyperglyce-