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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 63:669-677 (2008)
© 2008 The Gerontological Society of America

Tolerance and Efficacy of a New Enteral Formula Specifically Designed for Elderly Persons: An Experimental Study in the Aged Rat

Agathe Raynaud-Simon, Mirjam Kuhn, Julie Moulis, Julie Marc, Luc Cynober and Cécile Loï

1 Biological Nutrition Laboratory, Faculty of Pharmacy, Paris Descartes University, France.
2 Geriatric Department, Bichat University Hospital, Paris, France.
3 Clinical Chemistry Laboratory, Hôtel-Dieu Hospital, Paris, France.

Address correspondence to Agathe Raynaud-Simon, MD, PhD, Geriatrics Department, Bichat University Hospital, 46 rue Henri Huchard, 75877 Paris Cedex 18, France. E-mail: agathe.raynaud-simon{at}bch.aphp.fr


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
For the first time, a formula was specifically designed for the nutritional support of tube-fed elderly patients (Elderly-Specific Formula [ESF], Nestlé, Switzerland). It was tested against a standard formula (Sondalis Iso [SI], Nestlé Clinical Nutrition, Marne la Vallée, France) in sixteen 22-month-old Sprague Dawley rats fed by total continuous enteral infusion for 7 days. Body weight, stool weight, and nitrogen balance were measured daily. After death, muscle weight, plasma levels of amino acids, tissue protein, and amino acid content were measured. The ESF curbed weight loss, improved cumulative nitrogen balance, and increased jejunum protein content. Plasma levels of threonine, leucine, and isoleucine and the sum of total amino acids were higher in ESF-fed than in SF-fed rats. Threonine and isoleucine content in the soleus and gastrocnemius were higher in ESF-fed rats than SI-fed ones. ESF improved intestinal transit. Thus, in old rats, the ESF favored nutritional status more than a standard formula.

Key Words: Enteral nutrition • Whey protein • Nitrogen balance • Amino acids • Aged rat

AGING alters the control of metabolic homeostasis and generates specific nutritional requirements. More specifically, protein homeostasis in aging muscle is threatened by an imbalance between catabolism and anabolism: While there is no difference in terms of proteolysis, the anabolic response of muscle to nutritional stimuli is weaker than it is in younger persons (1–3). This contributes to a progressive loss of muscle mass (so-called "sarcopenia"), poor adaptation to food restriction (4,5), and a resistance to refeeding (6,7). Thus, although still a controversial point (8–10), protein requirements in terms of quantity are probably slightly higher in elderly than in young individuals (11). Furthermore, the quality of the proteins for protein metabolism appears to change during aging: As opposed to younger persons, protein gain is greater in elderly persons fed rapidly digested proteins (e.g., whey protein) than in elderly persons fed an isonitrogenous amount of slowly digested proteins (e.g., casein) (12). Aging is also associated with a decrease in insulin sensitivity (13); elderly populations are characterized by a high prevalence of impaired glucose tolerance (14) and an increased risk of developing diabetes with age, reaching 20% in persons older than 70 years (15).

Malnutrition is highly prevalent in geriatric populations, affecting as many as 4%–10% of community-dwelling elderly persons, 20%–40% of institutionalized persons, and 30%–70% of elderly hospitalized patients (16). Oral nutritional supplementation is usually efficient in improving nutritional status and outcome, but tube-feeding is recommended when food intake remains low or in patients presenting swallowing disorders (17). Elderly patients represent a large proportion of patients with home enteral nutrition (18). However, surprisingly, no specific enteral formula has yet been proposed for the elderly population.

Thus, a new formula was designed specifically for the nutritional support of tube-fed elderly patients (Elderly-Specific Formula [ESF], Nestlé, Switzerland). This formula is isoenergetic (100 kcal/100 mL) and slightly hyperproteinic (4 g of protein/100 mL, 16% of energy). The proteins used are mainly (2/3) whey proteins, which have been defined as "fast proteins" (19). To improve glycemic control, this formula supplies a moderately reduced quantity of carbohydrates (11 g/100 mL, 44% of energy) (20,21) and is supplemented with fructose (1 g/100 mL, 4.4% of energy) (22) and chromium (15 µg/100 mL) (23). The formula provides 40% of energy as lipids (4.5 g/100 mL); eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are supplied in amounts recommended for the elderly ({omega}6/{omega}3 ratio = 3.3) (11) because this population is known to have a limited desaturase activity (24). To improve both glycemic control and intestinal transit, the formula also incorporates fiber (1.4 g/100 mL, 55% insoluble and 45% soluble) (25,26).

We decided to compare this new ESF to a standard formula (Sondalis Iso [SI], Nestlé Clinical Nutrition, Marne la Vallée, France) in terms of protein metabolism, glycemic control, and tolerance.


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
Animal care complied with the French regulations for the protection of rats used for experimental and other scientific purposes (D 2001-486) and with all applicable European Community regulations (Official Journal of the European Community, L538 12:18:1986). Luc Cynober is authorized to perform experiments with rodents (authorization No. 75.461), and our team has the French government's authorization to perform surgery on rats (Christophe Moinard, authorization No. 75.522).

Sixteen 22-month-old male Sprague Dawley rats (579 ± 47 g, Charles River, L'Arbresle, France) were brought to our central animal facility and housed for a 2-week acclimatization period in individual metabolic cages that allowed the measurement of food intake and the collection of urine and feces. These cages were maintained at a constant temperature (21 ± 1°C) and humidity, on a 12-hour light/dark cycle. The rats were given free access to standard laboratory chow (16% protein, 3% fat, 60% carbohydrate, 12% water, vitamins, and minerals) supplying 290 kcal/100 g (A04; Safe, Villemoisson-sur-Orge, France), and water. During the first 2-week period (Figure 1), food intake was measured daily as was the weight of chow ingested (weight of chow given – weight of chow left in the bowl the day after). The rats ingested a mean of 23 ± 4 g/day of chow, that is, 67 kcal/day. This amount of energy intake became our reference for determining the nutritional goal for the period of enteral feeding.


Figure 01
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  		Figure 1. Experimental design. Gastrostomy was performed after a 2-week acclimatization period. Rats were allowed 1 week to recover from surgery before being randomized to receive either Sondalis Iso (SI) (Nestlé Clinical Nutrition, Marne la Vallée, France) or Elderly-Specific Formula (ESF) (Nestlé, Switzerland) in continuous enteral nutrition for 7 days. D = day

 
Surgical Procedure for Enteral Nutrition
On Day 7 (D7), a tube-feeding catheter was fitted. Surgical procedures were performed as described previously (27,28). After an overnight fast, the rats were anesthetized with isoflurane (3% in oxygen; Minerve, Esternay, France) and kept anesthetized by continuous isoflurane inhalation (1.5% in oxygen) throughout surgery. To prevent postsurgical dehydration, we performed subcutaneous infusion of saline (10 mL in the right and left side of the lower back) while the rats were anesthetized. The abdominal and neck areas were shaved, and the skin was carefully cleaned with polyvidone iodine (Betadine; Sarget Laboratory, Merignac, France). The animals then underwent a left-side laparotomy, and the greater curvature of the stomach was isolated. A polyethylene catheter (internal diameter 0.86 mm, external diameter 1.52 mm; Harvard Apparatus, Les Ulis, France) was placed inside the stomach and secured with a purse-string suture (using a 5.0 silk; Ethicon, Issy-les-Moulineaux, France). The catheter was tunneled subcutaneously to the neck and attached to a spring coil-swivel mechanism allowing the rat freedom of movement in the cage. The system was connected to a push-syringe (model 1140-101; Harvard Apparatus) for delivering the enteral nutrition. The rats received a single subcutaneous injection of analgesic approximately 20 minutes before the end of surgery (Temgesic, 0.05 mg/kg; Schering-Plough, Levallois-Perret, France) and were starved for the next 24 hours, being allowed only to drink.

Postoperative Care and Nutrition Program
The rats were allowed 1 week to recover from surgery (Figure 1). During this period, the rats had free access to the A04 standard laboratory chow and water. They were then randomized to be enterally fed with either SI or ESF. Then, enteral nutrition was progressively introduced on D0 (1 week after surgery) at a flow rate of 2 mL/hour for 3 hours, then 3 mL/hour for 16 hours, and finally settling at 3.5 mL/hour to supply 67 kcal/day, which corresponds to the spontaneous preoperative energy intake.

From D0 until the end of the experiment (D8), the enterally fed rats received only enteral nutrition (fed 24 hours a day at a constant infusion rate) but had free access to tap water. The two enteral formulas, ESF and SI, are isoenergetic and isovolumic (diet compositions are given in Table 1). The enteral formulas were diluted in water (80:20, vol/vol) to prevent clogging in the catheter.


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  		Table 1. Composition of the Diets (for 1 L, Supplying 1000 kcal).

 
Sample Processing
From D0 to D8 (Figure 1), the rats were weighed and urine collected daily using a container on a preservative (Amukin; Gifrer Barbezat, Decines, France). On D8, enteral nutrition was stopped, and the rats were immediately killed (i.e., at the fed state) by decapitation (after anesthesia with isoflurane). This time schedule was selected to assess the effect of the formulas on glycemic control.

Blood.-- Blood was sampled in heparin tubes and immediately centrifuged (10 min, 2500 g, 4°C). Part of the plasma was deproteinized (with sulfosalicylic acid, 30 mg/mL), and samples were stored at –80°C until amino acid analysis (29).

Liver.-- One piece of liver was promptly removed, weighed, frozen in liquid nitrogen, and stored at –80°C until analysis.

Muscles.-- The soleus, extensor digitorum longus, tibialis, and gastrocnemius were rapidly removed, weighed, frozen in liquid nitrogen, and stored at –80°C until analysis. These four muscles were selected because they differ widely in their fiber types and functions (30) and show different metabolic responses to stress (31,32).

Intestine.-- Ten centimeters of proximal jejunum and proximal ileum were promptly removed. These samples were washed with ice-cold 0.9% NaCl (wt/wt) through the lumen and inverted to collect the mucosa using glass scrapers. The intestinal mucosa was weighed, frozen, and stored at –80°C until analysis.

Parameters Studied and Analytical Methods
Urinary parameters.-- Urinary nitrogen was quantified by a pyrochemiluminescence-based method (Antek 7000; Antek, Houston, TX) (33). Nitrogen balance matched the difference between daily total nitrogen intake and daily total nitrogen urinary output. Cumulative nitrogen balance on D2 was expressed as nitrogen balance on D1 + D2; cumulative nitrogen balance on D3 was expressed as nitrogen balance on D1 + D2 + D3, and so on.

Creatinine was measured on an Olympus AU600 apparatus using the Jaffe method (34).

Myofibrillar protein degradation was evaluated by measuring excreted urinary 3-methyl histidine (3-MH) (35), which is released during myofibrillar protein breakdown without being either metabolized or reutilized for protein synthesis (36). In the rat, 3-MH is chiefly excreted in acetylated form. Samples were thus hydrolyzed with HCl (6 mol/L, vol/vol) at 100°C for 12 hours, then centrifuged (30 min, 2500 g, 4°C) and filtered on 0.20-µm filters before quantification by ion-exchange chromatography with ninhydrin detection (Hitachi L8500A; Tokyo, Japan) (37). Results are expressed in µmol 3-MH/mmol creatinine to factor muscle mass into the evaluation of myofibrillar protein breakdown.

Stools.-- Stools were collected and weighed daily; their consistency was recorded to evaluate the digestive tolerance to the formulas.

Tissue protein content.-- Muscle, liver, and intestinal protein content were determined using the method described by Fleury and Aberham (38), on a Genesys (Thermo Spectronic, Rochester, NY) spectrophotometer.

Plasma parameters.-- Plasma albumin and fibrinogen were assayed by immunonephelometry with an Array System 360 analyzer (Beckman Instruments, Gagny, France) and Beckman nephelometric-grade reagents for the quantitative determination in biological fluids. Rabbit immunoglobulin G (Dako, J2L Elitech, Labarthe Inard, France) cross-reacting with rat albumin was used for albumin determination. Rat albumin (Sigma-Aldrich, L'Isle d'Abeau, France) was diluted in buffer to obtain a calibration curve from 0.5 g/L to 5.0 g/L. Antiserum and plasma samples were diluted 10-fold in buffer prior to nephelometric assays (39). Results are expressed in grams per liter. Rabbit immunoglobulin G (Dako, J2L Elitech) crossreacting with rat fibrinogen was used for fibrinogen determination. Rat fibrinogen (Sigma-Aldrich) was diluted in 0.9% NaCl to obtain a calibration curve from 1.25 g/L to 10.0 g/L. Antiserum and plasma samples were diluted sixfold in buffer prior to nephelometric assays. Results are expressed in grams per liter.

Glycemia was measured via a hexokinase endpoint method at 340–380 nm (Olympus AU600; Rungis, France) (34).

Insulinemia was determined by radioimmunoassay using an INSIK-5 kit (Diasorin, Saluggia, Italy) and rat insulin (Linco, Saint Charles, MO) as a standard.

Plasma and tissue amino acid concentrations.-- Amino acid concentrations were determined by ion-exchange chromatography with ninhydrin detection (Jeol JLC-500V; Tokyo, Japan) (29). Our laboratory is registered under the European Quality Control Program (ERNDIM, Brussels, Belgium), thus guaranteeing the reliability of measurements for all the amino acids studied. Results are expressed in µmol/L of plasma, in µmol/g of tissue (liver), in µmol/ whole muscle, or in µmol/10 cm of intestine.

Statistical Analysis
Data are expressed as means ± standard error of the mean. Between-group comparisons were performed using a Student t test. When a parameter was measured several times during the study (body weight, daily energy and nitrogen intake, stool weight), the effect of time was analyzed using an analysis of variance (ANOVA) on repeated measures. All tests were performed at the 5% type I error level, using Statview software (Abacus Concepts, Berkeley, CA).


   RESULTS
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Enteral nutrition was well tolerated in all 16 rats (normal rat behavior and stools), and mortality was nil.

Nutritional Intake
From D1 to D7, the ANOVA on repeated measures showed no between-group differences in energy intake (data not shown). Nitrogen intake was slightly higher in ESF-fed than in SI-fed rats, but the difference was not significant (p =.08, Figure 2).


Figure 02
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  		Figure 2. Nitrogen intake during enteral nutrition, from Day 1 (D1) to D7, in Sondalis Iso (SI)-fed and Elderly-Specific Formula (ESF)-fed rats. Analysis of variance on repeated measures showed no between-group differences in nitrogen intake. D = day

 
Body Weight
The rats lost a mean of 17 ± 11 g/week during the initial 2-week acclimatization period. The week following gastrostomy, they lost another 43 ± 4 g. ANOVA on repeated measures showed a significant effect of time on body weight (p < 10–3) during these first 3 weeks. Then, when the rats were enterally fed, body weight remained stable in the ESF-fed rats (578 ± 21 on D8 vs 585 ± 23 g on D1, Student's paired t test, p = 0.4) whereas the SI-fed rats continued to lose weight (561 ± 19 on D8 vs 591 ± 24 g on D1, Student's paired t test, p =.003). Change in body weight was significantly different between groups (–7 ± 8 vs –30 ± 7 g, p =.04, Student's t test, Figure 3).


Figure 03
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  		Figure 3. Variation in body weight during enteral nutrition in Sondalis Iso (SI)-fed and Elderly-Specific Formula (ESF)-fed rats. The ESF-fed rats lost significantly less weight than the SI-fed rats did (Student's t test, *p =.04)

 
Muscle Weight
There were no significant between-group differences in weight of the extensor digitorum longus, soleus, tibialis, or gastrocnemius (Table 2).


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  		Table 2. Muscle Weights in Sondalis Iso* (SI)-Fed and Elderly-Specific** Formula (ESF)-Fed Rats.

 
Nitrogen Balance
The cumulative nitrogen balance on day 7 was significantly better in ESF-fed than in SI-fed rats (p =.02, Figure 4).


Figure 04
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  		Figure 4. Cumulative nitrogen balance from Day 1 (D1) to D7 in Sondalis Iso (SI)-fed and Elderly-Specific Formula (ESF)-fed rats. ESF-fed rats had a higher cumulative nitrogen balance on D7 than did SI-fed rats (Student's t test, *p =.02)

 
Myofibrillar Protein Degradation
The urinary 3-MH/creatinine ratio was, respectively, 35 ± 2 and 43 ± 3 µmol/mmol in SI-fed and in ESF-fed rats (mean values over the week of experiment). ANOVA on repeated measures showed no effect of time and no between-group differences for myofibrillar proteolysis.

Muscle, Intestinal, and Liver Protein Content
There were no statistically significant between-group differences in protein content of the extensor digitorum longus, soleus, tibialis, and gastrocnemius muscles (Table 3) and the liver. The protein content of the jejunum was significantly higher in ESF-fed than in SI-fed rats (p =.0008). In the ileum, the protein content was 40% higher in the ESF-fed group than in the SI-fed group, but the difference failed to reach significance.


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  		Table 3. Tissue Protein Content in Sondalis Iso{dagger} (SI)-Fed and Elderly-Specific Formula{dagger}{dagger} (ESF)-Fed Rats.

 
Muscle Amino Acid Content
Threonine and isoleucine content in the gastrocnemius and soleus muscles was higher in ESF-fed than in SI-fed rats (Table 4). Leucine and glutamate content in the gastrocnemius was also higher in ESF-fed than in SI-fed rats (Table 4). The soleus and gastrocnemius content in other amino acids was not different between the two groups (data not shown).


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  		Table 4. Muscle and Plasma Amino Acid Content in Sondalis Iso{dagger} (SI)-Fed and Elderly-Specific Formula{dagger}{dagger} (ESF)-Fed Rats.

 
Plasma Amino Acid Levels
Total plasma amino acid levels were higher in ESF-fed than in SI-fed rats (5415 ± 331 vs 3983 ± 316 µmol/L, p =.02). The plasma levels of threonine, leucine, and isoleucine were significantly higher in ESF-fed than in SI-fed rats (Table 4). There were no significant between-group differences in plasma levels of the other amino acids (data not shown).

Intestinal Amino Acid Levels
The jejunum and ileum contents in amino acids were not significantly different between groups (data not shown).

Plasma Albumin and Fibrinogen Levels
There were no statistically significant between-group differences in plasma levels of albumin (13.3 ± 1.3 and 12.2 ± 1.1 g/L for SI-fed and ESF-fed rats, respectively, p =.5) and fibrinogen (10.2 ± 0.8 and 12.9 ± 1.3 g/L for SI-fed and ESF-fed rats, respectively, p =.1).

Glycemia
There was no significant between group-difference in glycemia at death (7.2 ± 0.4 and 7.3 ± 0.3 mmol/L for SI-fed and ESF-fed rats, respectively, p =.9).

Insulinemia
There were no significant between-group differences in insulinemia (2.5 ± 0.2 and 2.7 ± 0.2 ng/mL for SI-fed and ESF-fed rats, respectively, p =.4).

Stools
ESF-fed rats had higher 7-day cumulative stool weight than SI-fed rats had (25 ± 2 g vs 9 ± 1 g, p =.0001). In addition, ANOVA on repeated measures showed that stool weight was significantly higher in ESF-fed than in SI-fed rats (Figure 5), with no effect of time. There was neither diarrhea nor loose stools in either group.


Figure 05
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  		Figure 5. Stool weight from Day 1 (D1) to D7 in Sondalis Iso (SI)-fed and Elderly-Specific Formula (ESF)-fed rats. Analysis of variance on repeated measures showed a group effect (p = 10–4) but no time effect (p =.19)

 

   DISCUSSION
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This is the first report on the tolerance and efficacy of an enteral formula developed specifically to improve protein metabolism, glycemic control, and intestinal transit in malnourished elderly persons. In this pilot study, focused on protein metabolism, glycemic control, and tolerance, we tested the new formula against a widely used standard formula (SI) in an original animal model of aged Sprague Dawley rats fed exclusively by continuous enteral infusion for 7 days. This original model of total continuous enteral nutrition in rats using a polymeric diet was developed previously in our laboratory (27,28). This model is closer to clinical situations than other models developed by other teams. To limit diet-related problems of high viscosity and consequent catheter obstruction, these teams gave a polymeric diet orally (ad libitum) (40–43) or by gavage (44,45), or administered elemental diet in enterally fed animals (46,47). For the purpose of the present work, our original model of total continuous enteral nutrition using a polymeric diet was adapted to old rats. The doses of analgesic and anesthetic were about half those used in younger rats when expressed as a function of body weight. Also, a subcutaneous infusion of saline during anesthesia was added to the protocol to prevent dehydration, because old rats spontaneously drink little in the 24 hours after surgery (Raynaud-Simon and Loï, January 2006, personal observation). Under these conditions of anesthesia and pain and nutrition management, the old rats were well able to cope with the gastrostomy surgery and the nutritional program. In addition, rats were allowed to recover for 1 week after surgery, being fed orally ad libitum, before enteral nutrition was started, to minimize the effect of surgery stress-induced catabolic losses.

The formula was specifically designed for elderly humans, but was tested in old rats. The mature rat laboratory diet we usually use provides 16% protein, 3% fat, and 60% carbohydrate (A04), but with no scientific evidence of this composition being optimal for a rat. In wild rats, the composition of the food supply may vary considerably. Thus, rats are liable to benefit from very different diet compositions. The findings of a positive nitrogen balance for both enteral formulas in our study do provide convincing evidence that the protein level was appropriate in this rat model.

We did not include a control group fed ad libitum orally after surgery to our experimental design because old rats that have endured surgery have a low spontaneous oral food intake, providing lower energy and protein than that of enterally fed rats, giving little relevance to comparisons.

Rats progressively lost weight when placed individually in a metabolic cage for 2 weeks before the gastrostomy, which suggests that old rats do not acclimatize well to a change in environment (48). However, this procedure was mandatory to measure energy intake, which was used afterward as a goal for enteral nutrition. The week after the gastrostomy (i.e., during the recovery period), the rats had lost more weight, which may be explained by both the surgical stress and the following 24-hour fasting period. After that, during the 7 days of enteral nutrition, the body weight of the ESF-fed rats did not vary significantly whereas the SI-fed rats lost further weight. Furthermore, the ESF-fed rats had a significantly better cumulative nitrogen balance than the SI-fed rats had. Nitrogen stool content measurement was not performed because it is well documented that nitrogen loss is mainly urinary and that nitrogen loss in the feces is constant in most clinical situations that do not imply bowel disease (49). Furthermore, stool weight was higher in ESF-fed than in SI-fed rats; if there was a difference in stool nitrogen excretion, it would have minimized the difference in cumulative nitrogen balance between groups.

These results suggest that the new formula is efficient in improving protein metabolism. The two formulas are isoenergetic, whereas the ESF provides a slightly higher quantity of nitrogen than the SI formula does (0.64 vs 0.61 g N/100 mL), although the actual amount of protein received was not significantly different between groups. Thus, this slight difference in the amount of nitrogen according to formula is unlikely to be an explanation for the efficiency of the new formula. More importantly, protein quality differed between the two diets. In the SI formula, half of the proteins were casein, whereas in the ESF, 2/3 of the proteins were whey proteins, which have a faster digestion rate than casein (the remaining proteins were soy proteins in both formulas). This finding probably explains why, being killed at the fed state, the ESF-fed rats had higher total plasma amino acid concentrations than SI-fed rats. This does not hamper the significance of our results, because protein metabolism occurs at the postprandial state and indeed, in elderly persons, in&!nbsp;contrast to young adults, whey proteins appear to be more beneficial to whole-body protein metabolism than casein, inducing protein gain as assessed by whole-body leucine balance (12).

The preferential sites of protein accretion in response to whey protein administration in the elderly population remain somewhat unclear, because splanchnic and muscle protein metabolism in humans or rats have never been studied simultaneously in response to various protein diets. Our results show that, in old rats, protein gain was directed mostly toward the intestine, and specifically to the jejunum, which is the part of the gut that plays the most prominent role in amino acid absorption. Additional studies considering mucosal cellularity would allow specification of the effects of the formulas on the intestinal structure. The age-related increase in splanchnic amino acid extraction, which has been described in both humans (50,51) and rats (52), may also partly explain this result. In these experiments, the increase in splanchnic extraction of both leucine and phenylalanine has been observed in old rats fed via a 1-hour continuous infusion in the duodenum or in elderly persons receiving repeated small oral boluses of 50 mL of liquid diet every 20 minutes for 4 hours, which somewhat mimics continuous enteral nutrition. Thus, we feel that the 24-hour continuous feeding paradigm we designed to feed the rats is likely to capture the differences in splanchnic extraction characterized in the elderly population and explain our results.

However, the ESF formula may also favor muscle protein metabolism: The protein content of the muscles was not significantly different between groups but, compared to the control diet, the ESF formula induced an increase in plasma total amino acid levels, plasma leucine levels, and plasma and muscle isoleucine and threonine contents. This finding is probably due to the difference in the amino acid pattern of whey proteins and casein, because whey proteins have a higher essential amino acid index and contain more leucine, isoleucine, and threonine than casein does (53). The increase in plasma total amino acid level appears critical in elderly persons, because it has been shown that impairment of protein synthesis in muscle from old rats and elderly people after meal ingestion (54,55) can be normalized by increasing peripheral availability of amino acids (56,57). Furthermore, essential amino acids drive muscle protein synthesis whereas non-essential amino acids have no effect in humans (58). Among the essential amino acids, branched-chain amino acids are important anabolic signals in the muscle (59). In particular, leucine at physiological concentrations stimulates protein synthesis by enhancing the sensitivity of muscle protein synthesis to insulin (60). In old rats, the blunted response of muscle protein synthesis to feeding can be restored by feeding a leucine-supplemented diet (61). Thus, the increase in plasma total amino acids in ESF-fed rats may promote protein metabolism both in the splanchnic area and in muscle. It is likely that the measurement of protein content was not sensitive enough to detect between-group differences. Further studies should focus on a direct assessment of protein synthesis (e.g., using the flooding dose method).

The response of albumin to feeding may have been blunted by inflammation, because the old rats presented high plasma fibrinogen levels with no difference between groups. Healthy old rats have been reported to have higher plasma fibrinogen levels than young rats (62), reflecting a low-grade inflammatory state. Moreover, our rats had a surgical gastrostomy, and surgery does induce an increase in fibrinogen levels in the rat (63). Thus, in our study, both aging and surgery explain the observed inflammation that may have inhibited a nutrition-related change in albumin levels. Similarly, in another study (64) on humans undergoing surgery, enterally fed patients had higher protein and energy intakes, better nitrogen balance, and fewer postoperative complications than did conventionally treated patients (intravenous fluids with nil by mouth), but there was no difference in the drop in albumin levels between the two groups. Disease-related inflammation is also often observed in malnourished elderly tube-fed patients; however, it will also be important to test ESF in medically stable, inflammation-free elderly patients fed enterally in the long run, to assess the efficacy of the formula to counteract the very slow catabolic losses that are linked to progressive aging. In addition, because this new formula contains more {omega}3 polyunsaturated fatty acids than a standard formula does, it will be of interest to evaluate more specifically its effect on inflammation and immunity in further studies.

Given that elderly populations are characterized by a high prevalence of limited glucose tolerance, the ESF formula was also designed to improve glycemic control in elderly patients (14). Furthermore, hyperglycemia is a well-known complication of enteral nutrition (65), occurring in as many as 34.5% of elderly tube-fed patients (66). However, in our study, glycemia in all rats appeared to be within the expected range for the postprandial state (67), with no between-group differences. Insulinemia was also not different between groups. The effect of this formula on glycemic control might be better assessed in hyperglycemic situations.

Stool weight was significantly higher in ESF-fed than in SI-fed rats. There was neither diarrhea nor loose stools, and weight of the stools remained lower than that of younger rats fed on laboratory chow (13 ± 7 g/day, unpublished data from our laboratory). Higher stool weight in ESF-fed rats is probably mostly due to the fiber content in the formula (68). Older individuals have a slower colonic transit than young individuals have (69), and constipation is one of the most frequent gastrointestinal symptoms in the elderly population, being reported by as many as 24%–40% of community-dwelling elderly individuals (70) compared with fewer than 2% of persons younger than 65 (71). Constipation also may be reported in up to 30% of tube-fed elderly patients (66). Our results suggest that the ESF formula may benefit intestinal transit by preventing or improving symptoms of constipation. Diarrhea is also a common complication of enteral nutrition. None of the rats in either group studied presented diarrhea, so we could not study the effect of the formula on that clinical issue.

Conclusion
ESF (a) curbed weight loss; (b) improved protein metabolism by increasing nitrogen balance, intestinal protein content, and amino acid availability; and (c) improved intestinal transit in continuously fed old rats in this pilot study. In addition, the protein composition of the formula induced modifications in plasma and muscle amino acid profiles that may foster protein synthesis. All these parameters are of importance when refeeding malnourished elderly persons, which is a population known (a) to have protein homeostasis threatened by imbalance between catabolism and anabolism, and as a consequence (b) to have specific protein quantity and quality requirements. Studies should now be conducted in elderly tube-fed patients to assess the value of this formula in clinical settings.


   Footnotes
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Decision Editor: Huber R. Warner, PhD

Received February 11, 2008

Accepted April 19, 2008


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Guillet C, Prod'homme M, Balage M, et al. Impaired anabolic response of muscle protein synthesis is associated with S6K1 dysregulation in elderly humans. FASEB J. 2004;18:1586-1587.[Abstract/Free Full Text]
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