APPLIED NUTRITIONAL INVESTIGATION Nutrition Vol. 14, No. 3, 1998

Dietary Peptides Improve Wound HealingFollowing Surgery

PAMELA R. ROBERTS, MD, KIMBERLY WARD BLACK, LATG, JEAN T. SANTAMAURO, MD,AND GARY P. ZALOGA, MD, FCCP

From the Department of Anesthesiology, Bowman Gray School of Medicine of Wake Forest University,Winston-Salem, North Carolina, USA

Date accepted: 14 July 1997

ABSTRACT

To determine if peptide-based enteral diets improve wound healing when compared to amino acid-based diets, aprospective randomized study was conducted using 38 male Sprague–Dawley rats. Following placement of a standardizedabdominal wound, 20 animals were randomized to an isonitrogenous peptide-based (PEP) versus amino acid-based diet(AA) for 10 d. In addition, 18 animals were randomized to an amino acid-based diet supplemented with the peptidecarnosine (CARN) or its constituent amino acids (Control). Diets were administered through small bowel feeding tubes.Wound bursting pressure was significantly higher in the PEP animals compared to the AA animals (1796 9 versus 138612 mmHg;P 5 0.02). In addition, wound bursting pressure was significantly greater in the CARN animals compared tothe Control animals (1436 10 versus 1166 8 mmHg; P 5 0.005). Peptide-based enteral diets improve wound healingwhen compared to nonpeptide generating amino acid-based diets. We also conclude that the dietary peptide carnosinerepresents a dietary peptide that improves wound healing when administered as part of a complete enteral formula. Thiseffect on wound healing may be clinically relevant because carnosine is not found in most enteral formulas.Nutrition1998;14:266 –269. ©Elsevier Science Inc. 1998

Key words: peptides, wounds, healing, carnosine, enteral, nutrition, surgery

INTRODUCTION

Unhealed wounds are a constant source of inflammatorymediators and a substrate for infection. As such, they prolongthe recovery of injured patients and may lead to multiple organfailure and death. Therefore, a major goal of nutritional supportof the injured patient is the improvement of wound healing.Numerous nutrients are known to be important for optimalwound healing and include total protein and calorie intake,1–5

arginine,1,6,7 zinc,1,8 ascorbic acid,1,9,10 and vitamin A.1,11,12

Early initiation of enteral nutrition following injury is alsoimportant for stimulating healing.13

Our laboratory has been studying the biogenic effects ofdietary amines.14 We have found that nitrogen utilization var-ies with the type of protein used to feed animals.15,16

We believe that peptides generated from dietary proteins dur-ing digestion play a role in optimizing growth and healing.These peptides may directly stimulate cells involved in the

growing or healing process or may act indirectly by augment-ing the production of growth factors. Our previous studiesassessed nitrogen utilization from dietary peptides using animalgrowth rates.15,16 Growth rates were higher in animals receiv-ing peptide-generating diets compared to amino acid-baseddiets.15

We now extend our previous results by assessing the bio-genic effects of dietary peptides on wound healing. We testedthe hypothesis that dietary peptides augment the healingof abdominal wounds when administered as part of a com-plete enteral nutrition program. We measured wound healingin animals randomized to a peptide- versus nonpeptide-gener-ating diet (i.e., amino acid-based diet) and in animals inwhich the dietary peptide carnosine was added to an aminoacid diet. Carnosine is a peptide that has been previouslyshown to augment wound healing following parenteraladministration.17,18

Supported by the Department of Anesthesiology, Bowman Gray School of Medicine, Winston–Salem, NC, USA.

Correspondence to: Pamela R. Roberts, MD, Dept. of Anesthesiology, Bowman Gray School of Medicine, Medical Center Boulevard, Winston–Salem, NC27157–1009, USA.

Nutrition 14:266–269, 1998©Elsevier Science Inc. 1998 0899-9007/98/$19.00Printed in the USA. All rights reserved. PII S0899-9007(97)00468-1

MATERIALS AND METHODS

This protocol was approved by our institutional animal careand use committee and followed National Research Councilguidelines for the care and use of laboratory animals. Healthy,well-nourished, male Sprague–Dawley rats (Harlan, Indianapolis,IN, USA) weighing 300–350 g were maintained on rat chow(Prolab Rodent Diet, Agway, Syracuse, NY, USA) prior to study.On the first day of study, animals were anesthetized with intra-muscular ketamine hydrochloride and intraperitoneal sodium pen-tobarbital. A 4-cm midabdominal incision was made beginningimmediately below the xyphoid process. A gastroduodenal feed-ing tube was inserted (into the third portion of the duodenum) andthreaded subcutaneously so that it exited through the back be-tween the scapula. The feeding tube was passed through a flexiblewire spring (which protected it from damage) and was securedwith suture to the skin. The abdominal wound was closed usingfour sutures placed uniformly in all animals (one at either end, twoequally spaced between the two end sutures). The skin was closedwith interrupted sutures. The spring and catheter were attached toan infusion swivel, which allowed the animals to move freely intheir cages during subsequent days of study.

Experiment 1

Immediately following surgery, animals were randomized toan amino acid-based diet (AA,n 5 10, VivonexTEN, SandozNutrition, Minneapolis, MN, USA) or a peptide-based diet (PEP,n 5 10, ReabilanHN, Nestle´, Deerfield, IL, USA). Animals wereadministered isonitrogenous quantities of the formulas (Table I).Formulas were administered to both groups of animals at a rate of4 mL/h. Because we matched formulas for nitrogen intake, caloricintake was slightly but significantly (P , 0.05) lower in the PEPgroup. Animals in the peptide diet group received 96 kcal/d and4.2 g protein/d while animals in the amino acid diet group received111 kcal/d and 4.2 g protein/d. The amounts administered havebeen previously reported to sustain growth15,16 in these animals.None of the animals were allowed oral intake during the study.Animals were maintained on the enteral feeds for 10 d, weighed,and given a lethal dose of pentobarbital, and the strength of theabdominal wound was measured using the bursting pressure tech-nique.13 The skin sutures were removed, exposing the healingabdominal wound. The retaining sutures in the abdomen werecarefully removed and a low pressure balloon was inserted into theabdominal cavity through a small transverse suprapubic abdomi-nal incision. The suprapubic incision was separate from the ab-dominal wound. The balloon was slowly inflated and the pressurerecorded when disruption of the wound occurred. This pressure iscalled the bursting pressure.13 The individual performing the

bursting pressure measurements was blinded to the dietary treat-ment.

Experiment 2

Following recovery from surgery, animals were randomized toisocaloric/isonitrogenous enteral diets (VivonexTEN) containingeither the dietary dipeptide carnosine (CARN,n 5 9) or an equalquantity of its component amino acids beta-alanine and histidine(Control,n 5 9). Both formulas were administered at a rate of 3mL/h and delivered 86 kcal/d and 3.9 g protein/d. No oral intakewas allowed during the study. Animals were sacrificed on theseventh postoperative day and wound strength measured using thebursting pressure technique described previously.

Data are presented as mean6 SEM and were analyzed usingpaired Student’s two-tailedt tests. AP value, 0.05 was consid-ered significant.

RESULTS

Experiment 1

All animals received the same quantity of formula per day.Protein intake was similar between PEP and AA groups (Table I).However, caloric intake was slightly but significantly (P , 0.05)lower in the PEP group (because formulas were matched fornitrogen intake). Weight gain was significantly greater in AA thanPEP animals (466 4 g versus 196 7 g over 10 d;P 5 0.01).However, abdominal bursting pressure was significantly greater (P5 0.02) in the PEP group compared to the AA group (Fig. 1).

Experiment 2

Both feeding groups (CARN and Control) received similarquantities of nutrition (Table II). Weight gain was similar for eachgroup (66 5 versus22 6 7 g over 7 d). However, the CARN-fedanimals had significantly (P 5 0.005) higher wound burstingpressures (Fig. 2).

DISCUSSION

The results of this study indicate that dietary peptides improvewound healing in rats. The first study compared two commercially

TABLE I.

EXPERIMENT 1: FORMULA COMPOSITION

PEP AA

Calories (kcal/mL) 1.0 1.16Protein (g/L) 44 44Arginine (g/L) 1.4 3.4Glutamine (g/L) 3.6 18.0Fat (g/L) 40 3.4Carbohydrate (g/L) 119 239Vitamin A (IU/L) 2656 2900Vitamin C (mg/L) 100 70Zinc (mg/L) 10 12

FIG. 1. Wound bursting pressure (mmHg) at 10 d in animals receiving PEPdiet versus AA diet. *P 5 0.02. PEP, peptide-based diet; AA, aminoacid-based diet.

PEPTIDES IMPROVE WOUND HEALING 267

available nutritional formulas that differed in the form of theirprotein. One contained amino acids (nonpeptide-generating diet)and the other contained peptides. However, these formulas alsodiffered in their composition of amino acids, lipid, and carbohy-drate. They were similar in their composition of minerals, traceelements, and vitamins. Both diets contained ample quantities ofessential fatty acids. To better determine if peptide compositionwas responsible for improved wound healing, we performed thesecond experiment where we compared two products that differedonly in the content of one peptide. This peptide was chosen forstudy because it had previously been shown to increase woundhealing when administered parenterally or intradermally.17,18Thepresent study is the first to demonstrate that the dietary peptidecarnosine improves wound healing when administered as part of acomplete enteral nutrition formula. It is also the first to demon-strate that a peptide-based enteral formula stimulates faster wound

healing compared to an amino acid-based (i.e., nonpeptide gener-ating) formula.

We and others have shown that body growth varies dependingon the form of protein fed to animals.15,16,19 Growth rates aregreatest in animals receiving peptide-based diets, intermediate inanimals receiving intact protein-based diets, and slowest in ani-mals receiving amino acid-based diets. These previous resultssuggest that the form of protein modulates nitrogen utilizationindependent of its nitrogen content. Possible explanations includestimulation of growth factors or improved utilization of nitro-gen.15,19,20 In support of the biogenic effects of peptides, wepreviously compared growth rates in animals receiving soy versuscasein protein. Each protein generates a different profile of pep-tides when digested in the gastrointestinal tract. Growth wassignificantly better in animals receiving soy protein.16 The currentresults also support our contention that peptides in the diet pro-duce specific biogenic actions independent of total nitrogen intake.

Nitrogen is absorbed from the gastrointestinal tract in theform of amino acids and peptides. None of our animals devel-oped diarrhea and stool quantity was grossly similar in allanimals. A previous study in rats demonstrated equal absorp-tion of animo acid, peptide, and intact protein diets.19 There-fore, we do not believe that differences in nitrogen absorptionexplain our wound healing results. However, peptides are ab-sorbed intact from the gastrointestinal tract21–23 and can pro-duce biologic effects.14,24 Therefore, it is plausible that thebiogenic effects of absorbed dietary peptides are responsiblefor the improved wound healing.

Carnosine (beta-alanine-histidine) is a dipeptide found in highconcentrations in muscle tissue (i.e., meat).25 Its specific roles inthe body are not known. However, carnosine is reported to possessantioxidant, pH buffering, immunostimulating, and neurotrans-mission properties. Previous studies indicate that carnosine im-proves wound healing when administered intraperitoneally17 orintradermally (into the wound).18 We now report that carnosinealso improves wound healing when administered enterally as partof a nutrition formula. Therefore, carnosine represents a dietarybiogenic peptide.

While timing of nutritional support, arginine and glutaminecontent, presence or absence of malnutrition, and content of mi-cronutrients have all been purported to affect wound healing, noneof these factors could explain the results of this study. Arginineand glutamine content were significantly lower in the peptidegroup (Table I), but this group demonstrated better wound healing.Diets in experiment 1 differed in lipid and carbohydrate content,but the preponderance of data would suggest that this was not afactor in specific bursting pressure and ultimate wound healingcapacity.

Our results and results from other studies indicate that dietarypeptides possess trophic actions within the body. The exact mech-anism for these trophic effects remain unclear. Dietary peptidesmay stimulate the production of growth factors such as growthhormone, insulin, or insulin growth factor 1 (i.e., IGF-1). In thisregard, we have previously found that IGF-1 levels in the bloodwere higher in animals receiving peptide-generating diets com-pared to amino acid-based diets.15 It is also possible that aminoacid entry into the cell via peptide transporters is more efficient forstimulation of protein synthesis than entry in the form of isolatedamino acids. Other possible mechanisms for the increased woundhealing with peptide versus nonpeptide generating diets includestimulation of collagen synthesis, increased blood flow to thewound, free radical scavenging, and generation of cytokine pro-files which better support wound healing.

The results of this study provide additional support for abiogenic action of dietary peptides. This and other studies indicatethat dietary peptides improve the utilization of nitrogen in the

FIG. 2. Wound bursting pressure (mmHg) at 7 d in animals receivingCARN diet versus Control diet. *P 5 0.005. CARN, carnosine added diet.

TABLE II.

EXPERIMENT 2: DAILY NUTRIENT INTAKE

CARN Control

Calories (kcal/mL) 1.2 1.2Protein (g/L) 54.0 54.0Arginine (g/L) 3.4 3.4Glutamine (g/L) 18.0 18.0Added carnosine (g/L) 10.0 0Added beta-alanine (g/L) 0 5.0Added histidine (g/L) 0 5.0Fat (g/L) 3.4 3.4Carbohydrate (g/L) 239.0 239.0Vitamin A (IU/L) 2900.0 2900.0Vitamin C (mg/L) 70.0 70.0Zinc (mg/L) 12.0 12.0

CARN, carnosine added diet.

PEPTIDES IMPROVE WOUND HEALING268

body as assessed by protein synthesis,20 nitrogen balance,19 bodygrowth,15,16,19 and now wound healing. We also report that thespecific dietary peptide carnosine improves wound healing when

administered as part of a complete enteral nutrition formula. Thiseffect may be clinically relevant because the carnosine content ofcurrent nutrition formulas is unknown.

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(For an additional perspective, see Editorial Comment on page 311.)

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