Effect of chain length on absorption of biologically active peptides from the gastrointestinal tract.

OBJECTIVES: Protein digestion generates many peptides in the gut lumen. Some of these peptides possess biological effects when tested using in vitro systems. It is clear that dipeptides and tripeptides can be absorbed intact from the gastrointestinal tract. However, the fate of larger peptides and small proteins remains unclear. Equally unclear are the biologic potencies of absorbed peptides and the quantity of peptide that must be administered into the gut to produce a biologic effect. Thus, the purpose of this study was to determine the effect of amino acid chain length on the ability of enterally administered peptides to produce biologic effects. METHODS: Small bowel feeding tubes, jugular catheters, and arterial lines were placed into adult male Sprague-Dawley rats. Rats were administered intravenous (50 microg) and enteral (125 and 500 microg) thyrotropin-releasing hormone (TRH, a tripeptide), intravenous (100 microg) and enteral (100 and 500 microg) luteinizing hormone-releasing hormone (LHRH, a decapeptide), and intravenous (0.5 mg) and enteral (0.5 and 25 mg) insulin (a 51-amino acid polypeptide). The quantity of peptide administered represented less than 0.5% of a rat's normal daily protein intake. The biologic effect of TRH, LHRH, and insulin were assessed using thyroid-stimulating hormone (TSH) response, follicle-stimulating hormone (FSH) response, and glucose. We also measured serum levels of insulin in the rats following enteral insulin administration. RESULTS: The results indicate that enteral TRH (125 and 500 microg) produced the same TSH response as intravenous TRH. The response to 500 microg enteral LHRH was 50% of the response to intravenous LHRH and the response to 25 mg enteral insulin was 30% of the response to 0.5 mg intravenous insulin. Serum insulin levels increased significantly following both 0.5 and 25 mg enteral insulin. CONCLUSIONS: These results support the concept that small (di- and tripeptides) and large (10-51 amino acids) peptides generated in the diet can be absorbed intact through the intestines and produce biologic effects at the tissue level. The potency of the enterally administered peptides decreases as the chain length increases. We postulate that absorbed dietary peptides play a role in the modulation of organ function and disease progression.

Dietary peptides improve wound healing following surgery.

To determine if peptide-based enteral diets improve wound healing when compared to amino acid-based diets, a prospective randomized study was conducted using 38 male Sprague-Dawley rats. Following placement of a standardized abdominal 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 peptide carnosine (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 (179+/-9 versus 138+/-12 mmHg; P=0.02). In addition, wound bursting pressure was significantly greater in the CARN animals compared to the Control animals (143+/-10 versus 116+/-8 mmHg; P=0.005). Peptide-based enteral diets improve wound healing when compared to nonpeptide generating amino acid-based diets. We also conclude that the dietary peptide carnosine represents a dietary peptide that improves wound healing when administered as part of a complete enteral formula. This effect on wound healing may be clinically relevant because carnosine is not found in most enteral formulas.

Enteral feeding improves outcome and protects against glycerol-induced acute renal failure in the rat.

Acute renal failure is a common cause of morbidity and mortality in critically ill patients and frequently results from vasoconstrictive ischemic injury to the kidney. Protein and amino acids can vasodilate renal blood vessels. Thus, we tested the hypothesis that enteral feeding could prevent renal ischemic injury using an experimental model in which renal vasoconstriction is believed to cause ischemic renal injury. This study was performed using male Sprague-Dawley rats, and renal injury was induced by glycerol injection into the hind limbs. The resulting muscle necrosis (rhabdomyolysis) causes acute renal injury. In the first part of the study, 35 animals were randomized to a peptide-based enteral diet or water via a duodenal feeding tube and subsequently injected with glycerol. Seventy-eight percent (14 of 18) of the animals receiving the enteral diet survived 3 d compared with 35% (six of 17) of the water-fed animals (p < 0.05). Blood urea nitrogen (47+/-8 versus 137+/-27 mg/dl) and creatinine (0.8+/-0.1 versus 2.0+/-0.3 mg/dl) were significantly lower in the enteral survivors than in the water survivors. In the second part of the study, renal plasma flow (para-aminohippurate clearance) and glomerular filtration rate (insulin clearance) were measured in similarly treated animals (n = 14) 1 d after injury. Renal plasma flow (4.83+/-0.65 versus 2.37+/-0.62 ml/min) and glomerular filtration rate (2.05+/-0.27 versus 0.89+/-0.22 ml/min) were significantly higher in the enteral group than in the water group. These data indicate that enteral feeding can prolong survival and decrease renal injury after glycerol-induced rhabdomyolysis. The mechanism for the protection is partly related to maintenance of renal blood flow.

Carnosine is a novel peptide modulator of intracellular calcium and contractility in cardiac cells.

Myocardial contractile failure is a common cause of morbidity and mortality in patients with ischemic heart disease and systemic inflammatory states such as sepsis. Accumulating evidence indicates that contractile failure is associated with dysregulation of myoplasmic calcium levels. In a search for biochemical causes for contractile dysfunction, we found that the dipeptide carnosine improves cardiac contractility and tested the possibility that carnosine plays a role in the regulation of intracellular calcium. Carnosine increased contractility in a dose-dependent manner (1-10 mM) in isolated perfused rat hearts. and it also increased free intracellular calcium levels in isolated myocytes. Carnosine increased myocyte tension via calcium release from the ryanodine receptor calcium release channel in skinned myocardial fibers and increased open-state probability and dwell time of the isolated ryanodine receptor calcium release channel in lipid bilayers. In addition. we report that carnosine sensitizes the contractile proteins so calcium. These results suggest a novel role for carnosine as a modulator of intracellular calcium and contractility in cardiac tissue.

Magnesium antagonizes the actions of lysophosphatidyl choline (LPC) in myocardial cells: a possible mechanism for its antiarrhythmic effects.

Patients with cardiac arrhythmias, ischemia, and infarction may benefit from administration of supplemental magnesium. However, the exact mechanisms for magnesium's beneficial effects remain unknown. Lysophosphatidyl choline (LPC), an amphipathic phospholipid released from cardiac cell membranes during ischemia, increases free intracellular calcium concentrations ([Ca]i) and has been implicated as a cause of cardiac arrhythmias and coronary artery spasm during myocardial ischemia. We postulated that magnesium acts by inhibiting cellular calcium overload induced by mediators such as LPC. Myocardial cells from male Sprague-Dawley rats were isolated from ventricular tissue samples and [Ca]i determined using the fluorescent dye, fura-2/acetoxymethyl ester, measured in a spectrofluorometer. The increase in [Ca]i after exposure to 100 and 200 microM LPC was recorded in cells suspended in modified Dulbecco's phosphate buffered saline solution with 0.2, 2.0, and 20 mM magnesium chloride. Differences were determined by analysis of variance with P < 0.05 considered significant. LPC significantly increased [Ca]i in the 100 microM (506 +/- 76 nM) and 200 microM (675 +/- 81 nM) concentrations, compared to baseline (301 +/- 25 nM). MgCl2 at both the 2.0 and 20 mM concentrations significantly blunted the increase in [Ca]i in myocardial cells exposed to LPC, whereas 0.2 mM MgCl2 was ineffective. LPC is a potent lipid mediator which increases myocyte [Ca]i in a concentration-dependent manner. Magnesium concentrations > or = 2.0 mM effectively antagonize the increase in [Ca]i induced by LPC. Thus, magnesium may limit intracellular calcium overload stimulated by ischemic-induced LPC release.

Enteral feeding minimizes liver injury during hemorrhagic shock.

Liver injury is common in patients following hemorrhage and sepsis. There are multiple etiologies for this liver injury which involve both decreased nutrient blood flow and direct cellular injury. Enteral nutrients vasodilate gut blood vessels and increase blood flow to the intestines and liver. Since enteral nutrients vasodilate gut blood vessels, we wondered whether luminal nutrition would prevent hepatic injury during shock states. We randomized Sprague-Dawley rats to saline or enteral nutrition via duodenal feeding tubes. Animals were then subjected to 60 min of hemorrhagic hypotension or intraperitoneal injection of lipopolysaccharide (LPS). Liver injury was assessed by measuring levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) before and after hemorrhage or LPS. Enteral nutrients significantly decreased liver injury following hemorrhage. AST increased from 246 +/- 17 to 1605 +/- 593 U/L in saline animals and 283 +/- 39 to 551 +/- 94 U/L in enterally fed animals. ALT increased from 60 +/- 4 to 726 +/- 355 U/L in saline animals and 61 +/- 6 to 161 +/- 38 U/L in enterally fed animals. Enteral nutrients did not significantly alter the increase in AST/ALT following LPS. These results indicate that enteral nutrients can decrease liver injury following hemorrhagic hypotension.

Gut bacterial translocation/dissemination explains the increased mortality produced by parenteral nutrition following methotrexate.

Mortality is reported to be higher in methotrexate (MTX)-treated animals receiving total parenteral nutrition (TPN), compared to animals receiving enteral nutrition. The increased mortality is felt to be related to gut atrophy and bacterial translocation. In this study, we examined the effect of MTX (30 mg/kg) on survival, body weight loss, gut mass, and gut bacterial translocation in rats randomized to TPN or enteral nutrition. Twenty-four male Sprague-Dawley rats (n = 8 per group) were randomized to TPN, a peptide-based enteral diet (PEP), or CHOW. Animals were weighed daily and followed for survival (6 days). A separate group of rats (n = 6 per group) were similarly randomized and sacrificed at 3 days. Mesenteric lymph node complex, liver, spleen, lung, and blood were cultured for translocating bacteria. Body weight loss was similar in all groups (12.0-16.9 g/day). Mortality was significantly (P < 0.05) higher in the TPN animals (100%), compared to PEP (50%) and CHOW (25%) fed animals. All tissues in the TPN animals contained large quantities of bacteria, while most tissues in the CHOW group were free of bacteria. Bacterial counts in the PEP tissues were intermediate between TPN and CHOW. There were no significant differences between groups for gut weights or mucosal protein content. This study supports a direct relationship between bacterial translocation and mortality in rats following MTX.

Human sepsis increases lymphocyte intracellular calcium.

OBJECTIVE: To determine whether free intracellular calcium is increased during human bacterial sepsis. DESIGN: Prospective controlled study of lymphocyte free intracellular calcium concentrations from patients with sepsis compared with critically ill nonseptic patients and healthy subjects. SETTING: A large multidisciplinary ICU of a university hospital. PATIENTS: Eleven patients with sepsis, six patients after cardiac surgery, six patients with head injury, and 22 healthy control subjects. INTERVENTIONS: Blood samples obtained for lymphocyte isolation and measurement of free intracellular calcium concentrations. MEASUREMENTS: Lymphocytes were isolated using Ficoll-paque centrifugation and free intracellular calcium concentrations were measured using the fluorescent dye fura-2. We also evaluated the effect of septic serum, endotoxin, tumor necrosis factor (TNF), and lysophosphatidylcholine on lymphocyte free intracellular calcium concentrations. MAIN RESULTS: Mean (+/- SEM) lymphocyte free intracellular calcium concentrations were significantly (p < .05) higher in the septic patients (176 +/- 12 nM) compared with cardiac surgical (112 +/- 9 nM), head-injured (110 +/- 11 nM), or healthy control patients (112 +/- 5 nM). Endotoxin (0.1 and 1.0 mg/mL) and TNF (10 and 100 ng/mL) did not alter lymphocyte free intracellular calcium values. Lysophosphatidylcholine (100 and 200 microM) significantly increased lymphocyte free intracellular calcium in a dose-dependent manner. Septic serum had no effect on resting lymphocyte free intracellular calcium concentrations but potentiated the free intracellular calcium response to lysophosphatidylcholine. CONCLUSIONS: Lymphocyte intracellular calcium homeostasis is altered during human sepsis. In addition, circulating factors present in septic serum are capable of altering cellular calcium handling.

Low dose calcium administration increases mortality during septic peritonitis in rats.

Calcium is essential for normal cellular function. However, increases in cell calcium are also involved in cellular dysfunction and death by activating destructive enzymatic processes such as proteases, nucleases, and lipases. Since ionized hypocalcemia is common during sepsis, calcium is often administered in an attempt to normalize circulating levels. Previous studies using endotoxin and large amounts of calcium demonstrated increased mortality in calcium-treated animals. This study extends these preliminary data to endogenous sepsis (cecal ligation) and low dose calcium therapy. Sprague Dawley rats (n = 9 per group) had jugular catheters placed and their cecums ligated. They were randomized to an infusion of D5W at 1 ml/hr, calcium chloride at 4 mg/ml/hr (Low Ca), or calcium chloride at 6 mg/ml/hr (High Ca) for 24 hr. An additional group of animals received calcium chloride at 6 mg/ml/hr but had no surgery. Animals were followed for 24 hr survival. A separate group of animals (n = 6 per group) was prepared in a similar manner. Blood was obtained 6 hr following cecal ligation for circulating endotoxin concentrations, and animals were sacrificed. Calcium infusion increased ionized calcium levels slightly (low Ca, 1.12 +/- .06 to 1.21 +/- .09; high Ca, 1.06 +/- .04 to 1.16 +/- .05; P less than 0.05). Survival (24 hr) was 100% in the nonseptic controls and D5W septic animals, 67% in the low Ca septic group, and 44% (P less than 0.05) in the high Ca septic group.(ABSTRACT TRUNCATED AT 250 WORDS)

Immediate postoperative enteral feeding decreases weight loss and improves wound healing after abdominal surgery in rats.

OBJECTIVE: To determine the effect of immediate vs. delayed (72 hrs) postoperative enteral feeding on weight loss and wound healing after experimental abdominal surgery. DESIGN: Prospective, randomized, controlled study. SETTING: Laboratory of a large university-affiliated medical school. SUBJECTS: Seventeen male Sprague-Dawley rats, each weighing 350 to 400 g. INTERVENTIONS: Four-centimeter longitudinal midabdominal incisions were made and gastroduodenal feeding tubes were inserted in the animals. The abdominal wound was closed in two layers. Immediately after closure, animals were randomized to receive immediate enteral feeding (early-fed group) with a peptide-based enteral formula or 5% dextrose in water at 4 mL/hr. Seventy-two hours after surgery, the 5% dextrose in water group was switched to the peptide formula (late-fed group). Animals were weighed daily. On postoperative day 5, the strength of the abdominal wound was determined using a balloon-bursting pressure technique. Blood was also obtained for measurement of insulin growth factor 1 concentrations. Mucosal protein content of the small bowel was measured. RESULTS: Total body weight loss was less in the early-fed group (26 +/- 4 vs. 46 +/- 5 g/5 days) and wound strength was increased in the early-fed group compared with the late-fed group (6 +/- 0.4 vs. 2.9 +/- 0.8 kPa; 45 +/- 3 vs. 22 +/- 6 mm Hg). There were no differences between groups for circulating insulin growth factor 1 concentrations or small intestinal mucosal protein concentrations. CONCLUSIONS: Immediate postoperative enteral feeding results in decreased weight loss and improved wound healing after abdominal surgery in rats.

Effect of rate of enteral nutrient supply on gut mass.

Early enteral feeding after injury is important for maintenance of gut integrity. However, enteral nutrients are frequently administered at low rates because of decreased gastrointestinal motility. These low rates are said to "maintain the gut." This study was performed to evaluate the effect of rate of enteral nutrient delivery on gut mass. Six male Sprague-Dawley rats had no surgery and served as controls (ad libitum rat chow diet). Twenty-four male Sprague-Dawley rats underwent abdominal surgery for placement of gastroduodenal feeding tubes (tip located 2 cm into intestine from pylorus) and were randomized (n = 6 per group) to ad libitum rat chow, 1/2 strength peptide diet (Reabilan HN, RHN) at 1 mL/h (1/2RHN-1 mL), full-strength peptide diet at 2 mL/h (RHN-2 mL), or full-strength peptide diet at 4 mL/h (RHN-4 mL). These diets supplied approximately 30%, 13%, 50%, and 100% of rat recommended daily allowances. The control animals gained weight (38 +/- 3 g over 5 days) whereas all postsurgery animals lost weight. Weight loss was greatest in the 1/2 RHN-1 mL (-55 +/- 3 g over 5 days) and RHN-2 mL (-52 +/- 6 g over 5 days) groups compared with the RHN-4 mL animals (-41 +/- 5 g over 5 days). All animals fed liquid enteral diets had reduced gut weights compared with chow-fed animals. Gut weights did not differ between control and postsurgery ad libitum chow animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Normal parathyroid hormone responses to hypocalcemia during cardiopulmonary bypass.

To determine whether the calcium-magnesium-parathyroid hormone-calcitriol (vitamin D) axis responds appropriately to the hypocalcemia that routinely follows initiation of cardiopulmonary bypass (CPB), we measured blood ionized calcium (CaI), total calcium (CaT), total magnesium (MgT), ultrafilterable magnesium (MgI), total protein, intact parathyroid hormone (PTH), and calcitriol concentrations at eight defined time points in 28 patients undergoing elective cardiac surgery. With the onset of CPB, CaI decreased from 1.14 +/- 0.02 to 0.91 +/- 0.03 mM, P less than 0.05) (n = 17), and then gradually returned to a normal value by the time of separation from CPB (0.98 +/- 0.01 mM). CaT, MgI, MgT, and total protein concentrations declined significantly upon initiation of CPB and remained depressed thereafter. PTH initially decreased upon initiation of CPB (from 50 +/- 8 to 24 +/- 9 pg/ml, n = 9, P less than 0.05), remained inappropriately decreased during the early phases of CPB, and then gradually increased to maximal concentrations in response to hypocalcemia (103 +/- 15 pg/ml) before emergence. Calcitriol concentrations (n = 8) were unchanged during surgery. Based on these initial results, which suggested an association between hypomagnesemia and the slow PTH response to hypocalcemia, measurements were repeated in 10 additional patients, to whom magnesium (Mg) (1 g MgSO4 in two separate intravenous doses) was administered. Mg administration neither altered the PTH response to ionized hypocalcemia nor hastened the return of CaI to normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Magnesium inhibits the hypertensive but not the cardiotonic actions of low-dose epinephrine.

Intravenous magnesium supplementation is often used to control cardiac arrhythmias and coronary artery vasospasm resulting from disturbances of magnesium homeostasis after coronary artery bypass surgery. Many such patients also require inotropic drug support of depressed myocardial function. However, increased serum magnesium concentrations directly depress cardiac contractility in animals and may interfere with catecholamine actions. To determine whether small intravenous doses of magnesium sulfate (MgSO4) interfere with the cardiotonic actions of epinephrine, we examined the hemodynamic effects of MgSO4 and epinephrine infusion in 17 cardiac surgical patients on their 1st postoperative day in a prospective, controlled study. In 11 patients, infusion of MgSO4 (7-mg.kg-1 bolus followed by 10 mg.kg-1.h-1 as a continuous infusion) increased serum magnesium concentrations by 44% (mean +/- standard error of the mean [SEM] of 0.8 +/- 0.1 to 1.2 +/- 0.1 mM; P less than 0.01) but had no significant effect on heart rate; mean arterial, central venous, or pulmonary arterial occlusion pressures; or cardiac output. Epinephrine infusion (30 ng.kg-1.min-1) significantly increased cardiac index (2.7 +/- 0.1 to 3.1 +/- 0.21.min-1.m-2; P less than 0.05); this effect was not altered by MgSO4 administration (n = 11). However, MgSO4 significantly blunted epinephrine's hypertensive action and prevented a significant increase in mean arterial pressure during concurrent MgSO4-epinephrine administration. Six placebo control patients were given two sequential infusions of epinephrine separated by a placebo infusion to rule out an effect of time on the hemodynamic response to epinephrine. Mean arterial pressure and cardiac index responses to epinephrine were identical before and after placebo infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Total parenteral nutrition increases mortality after hemorrhage.

OBJECTIVES: To determine the effect of total parenteral nutrition (TPN) and different enteral feeding formulas on survival and liver function following hemorrhage in rats. DESIGN: Prospective randomized controlled study. SETTING: Laboratory of a large university-affiliated medical school. SUBJECTS: Sixty-seven male Sprague-Dawley rats weighing 350 to 450 g. INTERVENTIONS: Jugular and gastroduodenal feeding catheters were inserted in animals 1 day before hemorrhage, and animals were started on one of six different fluid or nutritional regimens: TPN, iv saline, an enteral amino acid-based formula (AA) (Vivonex-TEN), an enteral peptide-based formula (PEP) (Reabilan-HN), an enteral intact-protein based formula (PRO) (Osmolite-HN), or enteral saline. A catheter was inserted in the tail artery and animals were hemorrhaged 5 mL/kg at baseline and 1 hr later. Animals were returned to their cages and observed for survival. Liver function was determined by measuring circulating bile acid levels at baseline and 24 hr after hemorrhage. MEASUREMENTS AND MAIN RESULTS: Mortality was significantly increased in animals receiving TPN (63%) and AA (24%). Mortality was 13% in animals receiving PRO and 0% in animals receiving PEP and saline. Liver function deteriorated in all animals after hemorrhage except the PEP group. CONCLUSIONS: TPN and AA increased mortality in animals after hemorrhage. PEP was associated with zero mortality and protection of liver function after hemorrhage.