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Original Research Communications |
| ABSTRACT |
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Objective: We tested the hypothesis that an uneven protein feeding pattern was more efficient in improving protein anabolism than was an even pattern.
Design: After a controlled period, 15 elderly women (mean age: 68 y) were fed for 14 d either a pulse diet (n = 7), providing 80% of the daily protein intake at 1200, or a spread diet (n = 8), in which the same daily protein intake was spread over 4 meals. Both diets provided 1.7 g proteinkg fat-free mass (FFM)-1d-1. Protein accretion and daily protein turnover were determined by using the nitrogen balance method and the end product method (ammonia and urea) after an oral dose of [15N]glycine.
Results: Nitrogen balance was more positive with the pulse than with the spread diet (54 ± 7 compared with 27 ± 6 mg Nkg FFM-1d-1; P < 0.05). Protein turnover rates were also higher with the pulse than with the spread diet (5.58 ± 0.22 compared with 4.98 ± 0.17 g proteinkg FFM-1d-1; P < 0.05), mainly because of higher protein synthesis in the pulse group (4.48 ± 0.19 g proteinkg FFM-1d-1) than in the spread group (3.75 ± 0.19 g proteinkg FFM-1d-1) (P < 0.05).
Conclusion: A protein pulse-feeding pattern was more efficient than was a protein spread-feeding pattern in improving, after 14 d, whole-body protein retention in elderly women.
Key Words: Aging protein feeding pattern fat-free mass nitrogen balance protein metabolism elderly women protein retention
| INTRODUCTION |
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A loss of protein necessarily involves an imbalance between protein synthesis and protein degradation. There is controversy as to whether whole-body protein synthesis and degradation, expressed per kg FFM, change with aging. In some experiments, no changes were found (47), whereas in others, protein turnover decreased with age (8). Furthermore, an alteration in muscle protein synthesis has been observed in the elderly compared with young adults (810). Such a decrease in protein synthesis could be involved in the age-related loss of muscle proteins.
Numerous experiments have shown that dietary protein intakes affect whole-body and muscle protein metabolism. High-protein diets in elderly people could increase nitrogen retention (1114). However, because of the methodologic limitations of the nitrogen balance method, this positive effect is debatable (15). In other experiments (16, 17), such diets increased whole-body protein synthesis in the postabsorptive state without significant effect on nitrogen balance. Studies in mature and old rats suggest that an increase in plasma amino acids is required in old animals for stimulation of muscle protein synthesis (18, 19). This was confirmed recently in elderly humans (20). However, high protein intakes could have deleterious effects on renal function in the elderly (21). One way to maintain the advantageous effects yet reduce the deleterious effects of high-protein diets would be to maintain the total daily protein intake but modulate the pattern of protein feeding during the day by using a pulse-feeding pattern. Interestingly, modifying the pattern of protein feeding by using slow (dietary amino acids are slowly absorbed) and fast (dietary amino acids are rapidly absorbed) proteins (22) or by using different meal patterns (eg, 3 discrete meals compared with continuous feeding) (23) was shown to modulate protein anabolism in young adults. A protein feeding pattern that combines meals rich and low in protein during the day may improve protein anabolism in elderly people by allowing for the advantages of both diets, ie, higher protein anabolism during postprandial periods and lower protein catabolism during postabsorptive periods (24). We tested this hypothesis by comparing 2 protein feeding patterns: 1) a pulse diet providing 80% of the daily protein intake in the midday meal and the remaining 20% in the morning and evening meals and 2) a spread diet in which the daily protein intake was provided fairly evenly over 4 meals fed over a 12-h period.
| SUBJECTS AND METHODS |
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Before the controlled dietary period, a dietitian met the subjects and customized a menu for each of them, taking care not to alter their dietary habits greatly (3 or 4 meals daily composed of usual food products). The diets were composed of ingredients selected from the following list: bread, sugar, jam, milk, butter, meat, gravy, fish, potatoes, vegetables, noodles, yogurt, cheese, juices, cakes, and fruit. All diets were provided by the experimental kitchen of the Human Nutrition Research Center. During the adaptive and experimental periods, all meals and drinks were served daily either at home or at the Human Nutrition Research Center and subjects were not allowed to eat or drink anything else except water, tea, or coffee (without sugar, milk, or any energy-containing substance). All subjects were motivated to participate in the study and were aware of the importance of compliance.
Because usual protein intake differed between the volunteers (0.81.6 g proteinkg body wt-1d-1), we used a 15-d adaptive period to achieve a similar protein status for all subjects. During this period, subjects received a controlled diet providing 1.2 g proteinkg FFM-1d-1 (ie, 0.74 gkg body wt-1d-1; Tables 2 and 3![]()
). The protein feeding pattern was similar to the subjects' usual feeding pattern, ie, the daily protein distribution was 10%, 60%, and 30% in the morning, noon, and evening meals, respectively. Energy intake was calculated by using dietary tables contained in the computer program GENI (Micro 6, Nancy, France) (Table 2
). During the following 14-d experimental period, 2 protein feeding patterns were compared. In one group (n = 7), most of the daily protein intake was provided in 1 of the 3 meals to provide a pulse feeding pattern: 79% of daily protein in the noon meal and the remaining 21% in the meals fed at 0800 (7%) and 2000 (14%). This was the pulse diet. In the other group (n = 8), subjects were fed 4 meals daily at 0800, 1200, 1600, and 2000 in which the protein content was spread out more evenly. This was the spread diet (Table 3
). The protein intake was greater during the experimental than during the adaptive period to promote an anabolic state. The increase in protein intake to 1.7 g proteinkg FFM-1d-1 (ie, 1.05 gkg body wt-1d-1) was compensated for by a decrease in carbohydrates, making the adaptive and experimental periods isoenergetic (Table 2
). During the experimental period, 70% of the protein was from animal products (dairy products, meat, and fish) and 30% was from plant products (cereals and legumes); the protein source was similar for both the pulse and spread diet groups. The daily energy repartition was similar between the 2 groups: 50.9 ± 1.4% of energy provided by the meal fed at 1200 in the pulse group and 48.6 ± 1.5% provided by the meals fed at 1200 and 1600 in the spread group (Table 3
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Nitrogen balance
Complete urine and feces collections were obtained daily during the last 5 d of the adaptive and experimental periods. After the total volume of urine was measured, aliquots were acidified with 6 mol HCl/L (50:1, by vol) and stored frozen. A fecal dye marker, 0.6 g red-carmine, was given orally at the start and end of each period of feces collection to identify the fecal samples for nitrogen analysis. After the feces were weighed, they were pooled. Duplicate meals were prepared and leftover food was collected for nitrogen analysis. All samples, excluding urine, were homogenized, and representative aliquots were stored at -20°C until analyzed further.
Aliquots of feces and meals (duplicates and leftovers) were freeze-dried for 48 h. The nitrogen content of aliquots of urine, feces, duplicate meals, and leftovers was measured by using an automated Kjeldahl method with a single-channel autoanalyzer (Kjeltec Auto 1030 Analyser; Tecator, Paris). Daily miscellaneous nitrogen losses were assumed to be 8 mgkg body wt-1d-1 (26). Nitrogen balance was calculated by subtracting the daily nitrogen losses from the daily nitrogen intake.
Urinary creatinine excretion was measured according to the Jaffé method with an autoanalyzer (Cobas Mira; Roche Diagnostic Systems, Neuilly sur Seine, France). Urinary creatinine excretion was stable throughout the experiment (data not shown), with a mean CV of 12%, suggesting complete urinary collection.
Protein turnover
Whole-body protein turnover was measured according to Waterlow et al (28). Two hundred milligrams [15N]glycine (99 atom%; Mass Trace Inc, Woburn, MA) was given orally to each subject on day 27 at 0800. On days 27, 28, and 29, urine was collected over two 12-h periods (from 0800 to 2000 and from 2000 to 0800). Twenty-fourhour nitrogen flux was determined from the urinary excretion of 15N in urea on days 2729. In addition, nitrogen flux was estimated from the urinary excretion of [15N]ammonia during the 12 h after [15N]glycine ingestion, which represents protein turnover during the fed state.
Urinary ammonia and urea concentrations were determined with enzymatic methods by using commercial kits (Ammonia kit 171-C; Sigma-Aldrich Chimie, St Quentin Fallavier, France). 15N enrichment of urinary ammonia and urea was measured by using a sodium-potassium form of a cationic ion-exchange resin that specifically binds ammonia from neutral solutions (29). The resin-ammonia complex was treated with alkaline hypobromite to produce a quantitative yield of molecular nitrogen, which was directly analyzed with a mass spectrometer (IRMS VG 903 E5; Micromass, Manchester, United Kingdom).
Nitrogen flux was calculated as described previously (28):
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The rates of protein synthesis and breakdown in the whole body were derived from the expression
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Statistics
Results are given as means ± SEMs. When comparisons were made between results obtained during the adaptive period and those obtained during the experimental period, a paired Student's t test was used because subjects acted as their own controls. Interactions between protein pattern (pulse or spread diet) and protein intake (adaptive or experimental period) were tested by two-way analysis of variance with repeated measures. The effect of diet on protein turnover (Z and B) was analyzed by using an unpaired Student's t test. The Statview program (version 4.5; Abacus Concepts, Inc, Berkeley, CA) was used for the statistical analyses.
| RESULTS |
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| DISCUSSION |
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In the anabolic state resulting from this increase in protein intake, the pulse protein pattern was more efficient at improving nitrogen balance than was the spread pattern. This was shown by 2 independent methods: FFM variations and nitrogen balance, with a significant, positive correlation between the 2 (r = 0.70, P < 0.005). This finding is consistent with the fact that the percentage of 15N not recovered in urine 3 d after ingestion of [15N]glycine was higher with the pulse than with the spread diet.
Feeding frequency (number of daily meals) was shown to modulate body composition and nitrogen retention (32), but the effects of protein feeding patterns on protein metabolism remain poorly studied and have given conflicting results. In young women, nitrogen balance was higher when the daily protein intake was spread fairly evenly over 3 meals than when spread over 2 meals, with 1 meal containing no protein (33); in young men, the same conditions resulted in no significant changes in nitrogen balance (34). Several differences between these studies, such as sex and age of the subjects, daily protein intake, and methods used, might explain the discrepancies. The present study provides the first evidence that at least in the short term in this group of elderly women, protein retention was more efficiently improved by a protein pulse feeding pattern than a spread feeding pattern.
Small discrepancies between some indexes used in the present study to assess the anabolic effects of protein intakes would not affect this conclusion. First, some subjects, mainly in the spread diet group, showed a decrease in FFM but were in positive nitrogen balance. This may have been because nitrogen retention was overestimated, which typically occurs with the nitrogen balance method, or because variations in FFM were measured over the whole experimental period (14 d), whereas nitrogen balance was determined on only the last 5 d of the experimental period. Nevertheless, because all determinations were made under the same experimental conditions, it was valid to compare the changes during the experimental period.
Second, the stimulation of protein anabolism that occurred during the experimental period corresponded to slight body weight losses. This was also observed by others (35). In fact, body weight losses were higher (40%) during the adaptive period than during the experimental period, but were not significantly different between the 2 groups. Thus, the women were in a more anabolic state during the experimental period than during the adaptive period. These small body weight losses may have been due to a slight energy deficiency because the women were fed 1.5 x their REE in accordance with the recommended dietary allowances for the elderly (26). However, Campbell et al (36) suggest that energy intake should be equal to 1.82 x REE to maintain energy balance, although other studies indicate that 1.5 (31) or 1.6 (13) x REE is sufficient to maintain body weight. The discrepancies between these studies may be a result of differences in physical activities of elderly subjects in free-living conditions. Nevertheless, because the energy contents of both diets were similar and because the effects of energy intakes on body weight were not significantly different between the 2 groups during the adaptive period, we conclude that the differences in body composition and nitrogen balance during the experimental period resulted from differences in the protein feeding patterns.
To obtain insight into the mechanisms involved in the regulation of protein balance, protein turnover was determined by using the end product method (ammonia and urea). Harmonic and arithmetic averages were not calculated because urea data represent whole-body protein turnover over the whole day, whereas ammonia data represent whole-body protein turnover in the fed state. In the present study, the nitrogen flux values, calculated from ammonia or urea data, were similar to those obtained by others (6, 30).
The protein pulse feeding pattern increased 24-h whole-body protein turnover. This effect was analogous to results obtained with an increase in protein intake (35, 37, 38). However, elderly women seemed to be less responsive to an increase in protein intake (17). The present study showed that the higher stimulation of daily protein synthesis with the pulse diet (19%) accounted for most of the increase in daily nitrogen balance and suggests that this pulse feeding pattern can improve the protein anabolic effect of an increased protein intake in elderly women.
In the fed state, the higher postprandial protein gain with the pulse diet resulted from slight alterations in whole-body protein turnover, ie, a small increase in protein synthesis (10%) and a small decrease in protein degradation (11%). We hypothesized that the protein feeding pattern might influence the diurnal protein cycle, which was described by Pacy et al (24). The increase in plasma amino acid and insulin concentrations that occurred after the midday pulse diet might have been responsible for the changes in protein synthesis and breakdown observed. Indeed, it is known that protein breakdown is inhibited by postprandial hyperinsulinemia (39) or a slight increase in amino acid concentrations (40) and that protein synthesis is stimulated by an even greater increase in amino acid concentrations (40, 41). Low-protein meals (eg, the pulse diet meals fed at 0800 and 2000) are known to induce modifications in protein turnover, especially by limiting postabsorptive losses (24). However, whether this occurred in the present study could not be verified because 24-h protein breakdown (urea data) values were higher during the pulse diet than during the spread diet, whereas they were similar between diet groups postprandially (ammonia data). This implies that postabsorptive protein breakdown was higher in the pulse than in the spread diet group.
In the present study, protein metabolism was measured in the whole body. However, responses of individual tissues to different diet patterns must be considered because of age-associated modifications in tissue protein metabolism. Muscle protein synthesis has been shown to decrease with aging (8, 9), but is still responsive to amino acid infusion (18, 20). However, in one study a high-protein diet failed to increase myofibrillar protein synthesis in the elderly (42). Thus, the higher protein retention seen with the pulse diet may have resulted from stimulation of muscle protein anabolism, although the increase in dietary amino acid extraction by splanchnic area (liver and gut) with aging (7) may limit this effect.
In conclusion, the 14-d pulse diet had a positive effect on the body composition of elderly women, particularly on FFM. It also resulted in higher nitrogen retention than did the spread diet because of higher whole-body protein turnover rates, particularly protein synthesis, over the whole day in the pulse diet group. Insofar as such results will be confirmed in a larger group of subjects, modulation of the pattern of protein feeding with respect to dietary habits (ie, the pulse diet in this study) may be a more attractive option than simply increasing the protein intake to improve protein turnover and protein retention in elderly women.
| ACKNOWLEDGMENTS |
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| FOOTNOTES |
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2 Supported by a PhD studentship to M-A Arnal and by grants from the Société Danone, Le Plessis-Robinson, France.
3 Address reprint requests to P Patureau Mirand, UEMA, INRA, Clermont-Ferrand-Theix, 63122 St Genès Champanelle, France.
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