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Articles

A Pancreatic Extract–Enriched Diet Improves the Nutritional Status of Aged Rats^{1 ,2}

Chantal Chambon-Savanovitch, Catherine Felgines, Stéphane Walrand, Francis Raul^*, Setareh Zarrabian, Marie-Thérèse Meunier, Marie-Chantal Farges, Luc Cynober and Marie-Paule Vasson³ ;

Laboratory of Biochemistry, Molecular Biology and Nutrition, Pharmacy School, EA 2416, Human Nutrition Research Center, Clermont-Ferrand, France; ^* CJF INSERM 95–09, IRCAD, Strasbourg, France; and INSERM, R. Debré Hospital, Paris, France

³To whom correspondence should be addressed. E-mail: M-Paule.VASSON{at}uclermont1.fr

	ABSTRACT

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Correction of the malnourished state, particularly common and severe in elderly people, is often unsuccessful. To improve the efficiency of realimentation, we evaluated the nutritional effect of a pancreatic extract (PE)-enriched diet in malnourished aged rats. Sprague-Dawley male rats were randomly assigned to 6 groups as follows: 1 group of control rats had free access to the diet for 12 wk (C group) and 5 groups were 50% food restricted for the same period. One food-restricted group was then killed (R group) and the 4 remaining groups were refed for 1 wk using a standard diet enriched either with two different doses of a pancreatic extract (2.4 or 4.8 g/d in PE₁ and PE₂ groups, respectively) or with an isonitrogenous casein hydrolysate (CH₁ and CH₂ groups, respectively). Profound alterations induced by food restriction (FR) were moderately corrected by refeeding, except nitrogen balance, which was reestablished in rats refed all diets (P < 0.01 vs. R). Supplementation of the food ration with a pancreatic extract clearly improved recovery. Indeed, body weight gain, both jejunal and ileal trophicity [jejunum: total height, PE₂: 849 ± 45 µm vs. CH₂: 768 ± 17 µm (P < 0.05); protein content, PE₂: 69.9 ± 5.7 mg vs. CH₂: 56.4 ± 4.8 mg (P < 0.01)] and nonspecific immune response in terms of H₂O₂ production by polymorphonuclear neutrophils and tumor necrosis factor (TNF-) by macrophages (PE₂, 20.7 ± 4.7 vs. CH₂, 8.7 ± 2.3, P < 0.05) were improved in rats fed PE₂. A pancreatic extract could improve the efficiency of realimentation in malnourished aged rats.

KEY WORDS: • rats • aging • malnutrition • casein hydrolysate • pancreatic extract

	INTRODUCTION

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Protein-energy malnutrition is common in elderly people, especially in aged hospitalized patients (Zawada 1996 ), in whom malnutrition is an important factor in morbidity and mortality. However, correction of the malnourished state is more difficult in elderly people than in younger subjects (Hébuterne et al. 1995 and 1997 ). One of the factors involved in the blunted response to nutritional support may be alterations in digestive processes. Human and experimental studies (Funakoshi et al. 1982 , Hollander and Dadufalza 1984 , Khalil et al. 1985 , Laugier et al. 1991 , Vellas et al. 1990 ) have described a decline in exocrine pancreatic function during aging. Although the exocrine pancreas has important reserves to maintain normal digestive capacity, malnutrition superimposed on the aging process may involve a slowing down of nutrient digestion (Al-Modaris et al. 1992 , Greenberg et al. 1988 , Miyasaka and Kitani 1989 ) and an intestinal atrophy, limiting absorption efficiency (Laugier et al. 1991 ). Intestinal alterations (Majumdar et al. 1997 ) are generally accompanied by protein metabolism disorders (El Haj et al. 1986 ) and impairment of the immune response (Chandra 1989 ). Hence an adequate nutritional intervention is necessary to counteract the disorders due to malnutrition. We hypothesized that pancreatic extracts, usually used as a palliative treatment of pancreatic insufficiency, could have a direct nutritional effect, improving the digestion and the absorption of the food ration, leading to more general effects on immunity, for example. Porcine pancreatic extract contains not only a variety of enzymes but other potential pharmacologic agents such as hormones and growth factors normally present in the exocrine secretion. We have previously shown (Farges et al. 1996 ) the value of providing a porcine pancreatic extract (Eurobiol^®, Parke Davis/Jouveinal Laboratories, Fresnes, France) during the refeeding of endotoxemic old rats. In this work, we focused on the efficiency of a pancreatic extract (PE)⁴on digestive structure and function, protein metabolism and nonspecific immune status during rehabilitation of malnourished aged rats.

	MATERIALS AND METHODS

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Animals.

Male Sprague-Dawley rats, 22 mo old (Iffa-Credo, L’Arbresle, France) were caged individually and kept at 23 ± 1°C in alternate 12-h light:dark cycles (dark from 0800 to 2000h). The laboratory was authorized by the French Ministry of Agriculture and Forestry to perform animal experiments and the NRC recommendations for the care and use of laboratory animals were followed.

Experimental design.

The rats were acclimated for 2 wk during which spontaneous food intake was measured daily. Mean food intake was 25.6 g/d during the second week of acclimatization. They were fed a standard nonpurified diet (A04, UAR, Villemoisson-sur-Orge, France) containing 170 g/kg proteins, 30 g/kg lipids, 587 g/kg carbohydrates and 210 g/kg water, fibers, vitamins and minerals. After the acclimation period, the rats were randomly assigned to 6 groups. One healthy control group had free access to the diet for 12 wk (C, n = 15). The others were food restricted for the same period, i.e., they were fed only 50% of the average food intake, thus receiving 12.8 g/d of the standard diet. At the end of the food restriction (FR) period, one group was killed (R, n = 14) and the four remaining groups were then refed the standard diet for 1 wk at 80% of the intake measured during the acclimatization period. The refeeding diet was enriched with either lyophilized porcine PE (Eurobiol) or casein hydrolysate (CH; UAR) used as an isonitrogenous control. The lyophilization process for PE preserves the hydrolytic activity of exocrine enzymes contained in the pancreas, e.g., lipase (100,000 IU), amylase (125,000 IU) and chymotrypsin (57,500 IU). Casein hydrolysate was obtained from hydrolysis into free amino acids (AA) (69%) and small peptides (31%) with an average molecular weight of 234 Da. Rats supplemented with the PE received 2.4 g/d (PE₁, n = 7) or 4.8 g/d (PE₂, n = 8) of Eurobiol powder, respectively. These doses were selected from previous experiments in rats (Farges et al. 1996 ). The corresponding isonitrogenous control groups received 1.6 g/d (CH₁, n = 7) and 3.2 g/d (CH₂, n = 8) of CH, respectively. Casein hydrolysate appears to be a suitable nitrogen placebo, i.e., a source of nitrogen devoid of any particular pharmacologic effect (Boza et al. 1996 , Poullain et al. 1989a ). The experimental conditions used, e.g., duration of restriction (Chambon-Savanovitch et al. 1999 ) and duration of refeeding (unpublished data), were based on results of preliminary experiments.

During the last 2 wk of the experiment, the rats were placed in individual metabolic cages. Urine was collected daily on an antiseptic solution (Amukine, Gifrer Barbezat, Décines, France) for the 2 d preceding the final day of experimentation, and then pooled, centrifuged and frozen at -80°C until analysis. Body weight was recorded daily throughout the experiment.

At the end of experimental periods and after overnight food deprivation, rats were anesthetized with diethyl ether and then killed by beheading between 0900 and 1100 h to minimize the influence of circadian rhythm on brush border enzyme levels (Saito et al. 1980 ). Blood samples were collected on calcium heparin (Léo, Saint-Quentin-Yvelines, France) to isolate white blood cells. The peritoneal cavity was washed with RPMI-1640 medium to isolate macrophages. The small bowel extending from the ligament of Treitz to the ileocecal junction was promptly resected and cut at its middle, yielding two portions, jejunum and ileum. A proximal 1-cm piece of each segment was removed for morphometry determinations. The next 20 cm of the jejunum and the ileum were divided into two equal segments, washed with ice-cold buffered 9 g NaCl/L and everted to scrape the mucosa. The first 10-cm segments of jejunum and ileum were used for hydrolase activity determinations and the second segments for protein content analysis. Skeletal muscles and the liver were also promptly removed to measure total protein content. The pancreas was removed to measure enzyme content. Tissues were rapidly frozen in liquid nitrogen and stored at -80°C until analysis.

	Analysis

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Nitrogen balance.

Total urinary nitrogen was quantified by chemoluminescence using an automatic apparatus (Model 7000N, Antek Instruments, Houston, TX) and used to calculate nitrogen balance, expressed in mg N/24 h, as the difference between daily nitrogen intake and daily urinary nitrogen excretion. Nitrogen in stools was considered to be negligible (<5%) and stable during starvation and refeeding in rats (Poullain et al. 1989b ); thus it was ignored here.

3-Methylhistidine/creatinine.

After deproteinization with sulfosalicylic acid (50 g/L), urine was diluted (1:1, v/v) in 12 mol/L HCl and incubated overnight at 95°C to deacetylate 3-methylhistidine (3-MH). After centrifugation (3500 x g for 10 min at 0°C), urine samples were filtered and stored at -80°C. 3-MH concentration was determined by ion-exchange chromatography with ninhydrin detection (model 6300, Beckman Instruments, Palo Alto, CA), after dilution of the sample within a lithium citrate buffer (pH 2.2) containing L--amino-ß-guanidino propionic acid as external standard (Sigma Chemical, Saint-Quentin Fallavier, France). Creati-nine was measured by the Jaffe reaction on a Hitachi 911 analyzer (Boehringer, Meylan, France).

Tissue protein content.

Skeletal muscles (soleus and tibialis anterior), liver and small intestine protein contents were determined as previously described (Farges et al. 1996 ).

Intestinal morphometry.

Villus height and crypt depth were measured as previously described (Raul et al. 1988 ). Total height was calculated as the sum of villus height and crypt depth.

Intestinal enzyme assays.

Sucrase and glucoamylase activities were determined by Dahlqvist’s modified technique (Cézard et al. 1979 ). Leucine aminopeptidase was measured as previously described (Ahnen et al. 1982 ). Enzyme activities were expressed as specific activities (mU/g protein).

Pancreatic enzyme assay.

A 100 g/L homogenate was prepared by homogenizing pancreas in ice-cold deionized water for 2 min under ice using an Ultra-Turrax T25 tissue crusher (Ika Labortecknic, Staufen, Germany). An aliquot of the homogenate was activated for 1 h at 30°C with 0.5% enterokinase (EC 3.4.21.9, Sigma-Aldrich, La Verpillère, France) in Tris-HCl buffer (0.1 mol Tris-HCl/L, 0.02 mol CaCl₂/L, pH 7.9). After centrifugation (3500 x g for 10 min at 0°C) of the activated mixture, supernatants were drawn off. Trypsin, chymotrypsin and elastase activities were then determined on supernatants by hydrolysis of N-benzoyl-Arg-p-nitroanilide, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and succinyl-Ala-Ala-Ala-p-nitroanilide, respectively. Working solutions of substrates (1 mmol/L) were prepared by diluting 200 mmol/L stock solutions in dimethyl sulfoxide with Tris-buffer. The increase in absorbance (410 nm) was followed at 30°C using a spectrophotometer (DU 640B, Beckman, Gagny, France) at intervals of 1 min for a period of 3 min. Activities are expressed as total activities per gram of pancreas [µmol/(min · g)].

Measure of oxidative response of stimulated polymorphonuclear neutrophils (PMN).

Leukocytes were isolated by gravity sedimentation using Plasmagel (Belon, Neuilly-sur-Seine, France) at room temperature for 45 min. Erythrocytes fell to the bottom of the tube and the supernatant containing granulocytes was drawn off. Residual erythrocytes were hemolyzed in a cold hemolytic solution (0.15 mol/L NH₄Cl, 0.1 mmol/L EDTA, 12 mmol/L NaHCO₃, pH 7.3). Cells were then washed twice with PBS+ (15 mmol/L PBS, 4 mmol/L MgCl₂ · 6H₂O, 4 mmol/L CaCl₂, pH 7.4) and adjusted to 10⁹ cells/L with RPMI-1640 medium (Sigma-Aldrich). Viability was controlled by Trypan blue exclusion test. PMN (10⁹ cells/L) were preincubated for 15 min with 5 µmol 2'7'-dichlorofluorescin diacetate/L (DCFH-DA, Acros Organics, Noisy Le Grand, France) in a water bath with horizontal agitation at 37°C. DCFH-DA diffuses into the cells and is then hydrolyzed to 2'7'-dichlorofluorescin (DCFH). Cells were stimulated by 10^-6 mol/L phorbol myristate acetate (PMA) for 10 min at 37°C to induce H₂O₂ production. The H₂O₂ produced caused the oxidation of nonfluorescent intracellular DCFH to highly fluorescent dichlorofluorescein (DCF). PMN were discarded and DCF fluorescence was measured using flow cytometry analysis (Epics XL, Beckman Coulter, Villepinte, France). Results were expressed as the ratio of fluorescence of PMA-stimulated PMN to fluorescence of nonstimulated PMN. For technical reasons, this assay was not performed in the CH₁ and PE₁ groups.

TNF- production by stimulated peritoneal macrophages.

Cell suspensions were sedimented at 1300 x g/min for 10 min at 4°C. Viability of cells was controlled using the Trypan blue exclusion test, and cells were counted and adjusted to a concentration of 10⁹ cells/L in RPMI-1640 medium supplemented with fresh L-glutamine (2 mmol/L, Sigma-Aldrich), penicillin (100,000 U/L, Sigma-Aldrich), streptomycin (100 mg/L, Sigma-Aldrich) and 10% fetal calf serum (Sigma-Aldrich). Macrophage suspension was distributed as 1 mL/well in multiwells (Falcon, Lincoln Park, MI), and macrophages were selected for their ability to adhere to a solid surface when incubated at 37°C for 2 h in a 5% CO₂ humidified atmosphere. Adherent cells were then washed 3 times using RPMI-1640 medium. For individual rats, each well was supplemented or not with a lipopolysaccharide solution at 40 mg/L (LPS from Escherichia coli serotype 0127: B8, Sigma-Aldrich) and multiwells were incubated again for 3 h in the conditions described above. Culture media were then collected and release of TNF- by macrophages was measured by ELISA (Factor test X^TM rat TNF- ELISA kit, Genzyme, Cergy Saint-Christophe, France). Results were expressed as the ratio of TNF- produced by LPS-stimulated macrophages to TNF- produced by nonstimulated cells. For technical reasons, this assay was performed on the R, CH₂ and PE₂ groups.

Statistical analysis.

Results are expressed as means ± SEM. Statistical analysis was performed with a one-way ANOVA followed by a Newman-Keüls test using a PCSM software package (Deltasoft, Meylan, France). All significant differences (P < 0.05) obtained are presented, except those between CH₁ and PE₂ groups and those between CH₂ and PE₁ groups. Correlations were performed using Pearson’s test.

	RESULTS

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Nutritional status and protein metabolism

    Body weight evolution. Throughout the experiment, control rats presented stable body weight (719 ± 10 and 734 ± 10 g at the beginning and end of the experiment, respectively). The 12-wk FR produced an average body weight loss of 43%. During refeeding, rats gained body weight. A higher rate of weight gain, expressed as the percentage of body weight at the end of the FR period, was observed in PE₁ rats (12.8 ± 1.6%) compared with those of the CH₁ group (5.4 ± 2.3%, P < 0.05). Weight gains in PE₂ (16.3 ± 2.8%) and CH₂ (11.5 ± 1.4%) groups were not different.

    Nitrogen balance (Fig. 1 ). Nitrogen balance fell dramatically after FR (P < 0.01) and was completely restored by refeeding all diets (P < 0.01 vs. R).

View larger version (46K): [in this window] [in a new window]   Figure 1. Nitrogen balance in aged rats with free access to a standard diet (C); or 50% food restricted for 12 wk with a standard diet and then killed (R); or food restricted and then refed a standard diet enriched with 2.4 g/d (PE1) or 4.8 g/d (PE2) pancreatic extract, or with 1.6 g/d (CH1) or 3.2 g/d (CH2) isonitrogenous casein hydrolysate for 1 wk. Results are expressed as the difference between daily nitrogen intake and daily urinary nitrogen excretion. Values are means ± SEM, n = 7–15. Means with no common letters differ, P < 0.05.

    Tissue weight and total protein content. The skeletal muscle weight and protein content were not significantly modified by FR or by refeeding, except for tibialis anterior protein content which was greater in the C group than in the CH₁ group (Table 1 ). Splanchnic tissue weight and total protein content were significantly lowered by FR (P < 0.01, Figs. 2 and 3). During refeeding, liver weight and protein content were improved in the PE₁, CH₂ and PE₂ groups compared with the R group (P < 0.01) although they did not reach control values (P < 0.01, Fig. 2 ). The PE₁ and CH₂ groups presented a higher protein content than the CH₁ group. Jejunal mucosa weight and protein content were restored by refeeding (Fig. 3A and B). These variables were higher in the PE₂ group compared with the others, including the C group (P < 0.01). In the ileum, only the highest dose of PE restored both mucosa weight and protein content (Fig. 3C and D )_. The weights of liver, jejunal and ileal mucosa were strongly correlated with their respective total protein contents (liver, r² = 0.92, P < 0.0001; jejunum, r² = 0.95, P < 0.0001; ileum, r² = 0.90, P < 0.0001).

View larger version (24K): [in this window] [in a new window]   Figure 2. Liver weight (panel A) and protein content (panel B) in aged rats with free access to a standard diet (C); or 50% food restricted for 12 wk with a standard diet and then killed (R); or food restricted and then refed a standard diet enriched with 2.4 g/d (PE1) or 4.8 g/d (PE2) pancreatic extract, or with 1.6 g/d (CH1) or 3.2 g/d (CH2) isonitrogenous casein hydrolysate for 1 wk. Values are means ± SEM, n = 7–15. Means for a variable with no common letters differ, P < 0.05.

View larger version (41K): [in this window] [in a new window]   Figure 3. Small intestine mucosa weight and protein content in aged rats with free access to a standard diet (C); or 50% food restricted for 12 wk with a standard diet and then killed (R); or food restricted and then refed a standard diet enriched with 2.4 g/d (PE1) or 4.8 g/d (PE2) pancreatic extract, or with 1.6 g/d (CH1) or 3.2 g/d (CH2) isonitrogenous casein hydrolysate for 1 wk. Panels A and C: jejunal and ileal mucosal weights, respectively; panels B and D: jejunal and ileal mucosa protein content, respectively. Values are means ± SEM, n = 7–15. Means for a variable with no common letters differ, P < 0.05.

    Intestine morphometry. FR produced a significant drop in jejunal (P < 0.05) and ileal (P < 0.01) total heights (Fig. 4 ). Refeeding restored jejunal total height in rats fed all diets. In addition, jejunal total height was greater in the PE₂ group than in the other groups including the C group, resulting in an increase in both villus height and crypt depth. In the ileum, total height was recovered only in the PE₂ group due to increases in both villous height and crypt depth.

View larger version (36K): [in this window] [in a new window]   Figure 4. Intestinal morphology in aged rats with free access to a standard diet (C); or 50% food restricted for 12 wk with a standard diet and then killed (R); or food restricted and then refed a standard diet enriched with 2.4 g/d (PE1) or 4.8 g/d (PE2) pancreatic extract, or with 1.6 g/d (CH1) or 3.2 g/d (CH2) isonitrogenous casein hydrolysate for 1 wk. Total height is the sum of villus height and crypt depth. Values are means ± SEM, n = 7–15. Means for a variable with no common letters differ, P < 0.05.

    Measure of oxidative response of stimulated PMN. H₂O₂ production by stimulated PMN was significantly decreased by FR (Fig. 5 , P < 0.01). Refeeding led to a partial recovery of immune response by stimulated PMN in the PE₂ group (+76% vs. R group, P < 0.05).

View larger version (23K): [in this window] [in a new window]   Figure 5. H2O2 production by stimulated polymorphonuclear neutrophils (PMN) in aged rats with free access to a standard diet (C); or 50% food restricted for 12 wk with a standard diet and then killed (R); or food restricted and then refed a standard diet enriched with 2.4 g/d (PE1) or 4.8 g/d (PE2) pancreatic extract, or with 1.6 g/d (CH1) or 3.2 g/d (CH2) isonitrogonous casein hydrolysate for 1 wk. Results are expressed as the ratio of fluorescence of PMA-stimulated cells to fluorescence of nonstimulated cells (AU, arbitrary units).Values are means ± SEM, n = 7–15. Means with no common letters differ, P < 0.05.

    TNF- production by stimulated peritoneal macrophages.

	DISCUSSION

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Results of preliminary experiments showed that a 12-wk 50% FR period induces severe malnutrition in aged rats and that most damage occurred in the splanchnic area rather than in peripheral tissues (Chambon-Savanovitch et al. 1999 ). Thus, protein-energy malnutrition, such as that produced in our study, induced multiple changes in aged rats, including a strong reduction in body weight and an impairment of intestine morphology and function, as previously described in young animal and human models of starvation (Gorostiza et al. 1985 , Waterlo and Stephen 1968 ). However, the response to refeeding seems to differ according to the age of the population studied (Hébuterne et al. 1995 and 1997 ). Because pancreatic secretions are important for intestinal trophicity and proliferation (Vellas et al. 1990 ), we hypothesized that short-term administration of PE could help to promote realimentation in malnourished aged rats.

Our results showed that body weight was strongly affected by FR, consistent with the results of Barrows and Roeder (1965) and suggesting an important loss of body fat mass. Simultaneously, nitrogen balance fell dramatically, mainly as a result of the drop in nitrogen intake. Our results reflect a defect of adaptation of aged rats to FR as previously described (Felgines et al. 1999 ). Refeeding with PE improved body weight gain from the lowest dose. In terms of nitrogen balance, refeeding completely restored nitrogen accretion in rats fed all diets.

In the splanchnic area, FR induced a decrease in tissue weight that was closely correlated with a marked loss in total protein content. In particular, FR caused jejunal and hepatic protein depletion, when expressed as mg protein/g tissue (data not shown), reflecting the sacrifice of visceral proteins, whereas skeletal muscle proteins were preserved, in agreement with the literature (Goodman and Ruderman 1980 , Holecek et al. 1995 ). Correspondingly, both muscle protein content and the 3-MH/creatinine ratio, usually used as a marker of proteolysis, were unmodified by the treatments, indicating the sparing of myofibrillar proteins. In the small intestine, FR-induced alterations may be related to the mucosal atrophy resulting from a decrease in both villus height and crypt depth, thus reflecting a diminution of both cell maturation and renewal, respectively. These results suggest an impairment of the intestinal structure during FR in aged rats. In our study, a CH-enriched diet induced an incomplete recovery of ileal structure, reflecting the difficulty in correcting the malnourished state in aged rats. However, our results show that refeeding a diet supplemented with PE improved the structure of the intestine, suggesting that it could promote the adaptative response to refeeding in malnourished aged rats. During FR, structural modifications were accompanied by a reduction of digestive capacities of the intestine, as shown by the lowered activity of hydrolases, which may result from both mucosal atrophy and specific effects caused by the limitation of their substrates (Reville et al. 1991 ). Globally, refeeding only partially restored hydrolase activities in rats fed the various diets. Similarly, Reville et al. (1991) found in starved aged rats that refeeding produced a partial recovery, whereas it induced a quick repair of digestive function in young rats. The pancreas contents of exportable trypsin and chymotrypsin were elevated more after refeeding, suggesting that the synthesis and storage capacities of the gland were improved. This might be followed by increased secretion of pancreatic enzymes in the intestine, contributing to the observed improvement of intestine trophicity, especially when PE was supplemented.

Together with impairment of the small intestine, FR induces abnormalities of the nonspecific immune status. Lesourd and Mazari (1997) showed an alteration of PMN and macrophage functions in malnourished elderly people. Here, we show that the nonspecific immune system was affected by FR in terms of H₂O₂ production by activated PMN, reducing the oxidative capacity of these cells and suggesting a decrease in bactericidal functions in aged rats. This does not seem specific to aging because Lipschitz and Udupa (1986) reported a decreased bactericidal activity in undernourished adult animals. Although refeeding has been shown to improve specific immune response in malnourished elderly persons (Lesourd and Mazari 1997 ), data on nonspecific immune response are scarce (Ortega et al. 1993 ). In our study, refeeding with a high dose of PE improved H₂O₂ production by stimulated cells. In addition, Munoz et al. (1994) showed that malnutrition is also associated with altered monocyte functions, including a decrease in cytokine secretions in animals and patients. Moreover, these authors reported an improvement in cytokine production (interleukin-1ß, TNF-) by monocytes with nutritional rehabilitation in malnourished infants. In our study, refeeding with a high dose of PE increased TNF- production by stimulated macrophages. Taken as a whole, our data indicate that PE may improve the nonspecific immune response.

In conclusion, this study provides new data on the effect of a realimentation enriched with PE in chronically malnourished aged rats. Taken as a whole, our results indicate that aged rats exhibit a defect of adaptation to a long-term severe FR, and the resulting impairment is incompletely corrected by a standard realimentation. In this context, a short-term supplementation of the food ration with PE improved the recovery. Body weight gain, small intestinal trophicity and nonspecific immune status were markedly improved in rats receiving the pancreatic extract. Hence, a pancreatic extract improves the efficiency of realimentation in malnourished aged rats. Whether the results of this study can be extended to humans remains to be determined. Lesourd et al. (1997) showed previously that a PE-enriched diet increases albuminemia in malnourished elderly people. Other study must be conducted to confirm these results.

	ACKNOWLEDGMENTS

 
We are indebted to J. Chassagne (CJP Hospital) for helpful collaboration. We thank P. Davot (Laboratory of Biochemistry, Molecular Biology and Nutrition), P. Rousset (Laboratory of Human Nutrition) and J. Macry (INSERM, R. Debré Hospital) for their expert technical assistance and S. Allouche (Jouveinal Laboratories) for interesting discussions throughout the study.

	FOOTNOTES

 
¹ Presented as an oral communication at the 14th meeting of the Club Français du Pancréas, October, 1998, Saint-Malo, France (Savanovitch, C., Felgines, C., Walrand, S., Raul, F., Cézard, J.-P., Allouche, S., Cynober, L. & Vasson, M.-P. Efficacité d’un extrait pancréatique comme starter de la réalimentation chez le rat âgé dénutri).

² Supported in part by a grant from EURORGA Laboratories, Jouveinal Parke-Davis group (Fresnes, France).

⁴ Abbreviations used: AA, amino acids; C, group fed a standard diet; CH, casein hydrolysate; CH₁, group refed a standard diet supplemented with 1.6 g/d CH; CH₂, group refed a standard diet supplemented with 3.2 g/d CH; DCF, dichlorofluorescein; DCFH, dichlorofluorescin; DCFH-DA, dichlorofluorescin diacetate; FR, food restriction; LPS, lipopolysaccharide; 3-MH, 3-methylhistidine; PE, pancreatic extract; PE₁, group refed a standard diet supplemented with 2.4 g/d PE; PE₂, group refed a standard diet supplemented with 4.8 g/d PE; PMA, phorbol myristate acetate; PMN, polymorphonuclear neutrophils; R, group fed a standard diet (50% food restricted) for 12 wk and then killed; TNF-, tumor necrosis factor .

Manuscript received March 15, 2000. Initial review completed June 27, 2000. Revision accepted December 6, 2000.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Analysis RESULTS DISCUSSION REFERENCES

 

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