© Borgis - New Medicine 2/2006, s. 43-47
Joanna Myszkowska-Ryciak, Janusz Keller, Jacek Bujko
The influence of feeding frequency on dietary protein utilization – a model study
Faculty of Human Nutrition and Consumer Sciences Department of Dietetics Warsaw Agricultural University, Warsaw, Poland
Head of Department: Prof. Joanna Gromadzka-Ostrowska, MD, PhD
Aim: The purpose of the study was to examine the influence of feeding frequency on dietary protein utilization during growth.
Material and method: A model study was performed on 36 growing, female Wistar rats. Animals were randomly divided into 2 groups (A, B) and maintained in individual cages in controlled conditions with free access to water. After acclimatization each rat from group A was paired with corresponding animal from group B (weight criterion). In pairs animals were fed with the same amount of commercial rats´ diet divided into equal meals: 2 (rat A) or 4 (rat B). Twenty-four hours urine collections were performed twice for each pair of rats: on day 11 and 22; body weight was controlled every day. Starting from day before urine collection all animals received 15N glycine (0.5 mg/g feed) in each meal. After 25 days rats were killed and organs: liver, heart, kidney, small intestine, muscles: gastrocnemius and soleus were excised. Dietary protein utilization was characterized by urine total nitrogen (urea nitrogen and 15N) excretion. Additionally body weight, body chemical composition and organs weight were analyzed.
Results: Statistical analyzes showed no differences in body weight, body chemical composition and organs weight between animals fed 2 and 4 meals a day. There were no differences in urine parameters (total nitrogen, urea nitrogen and 15N).
Conclusions: Feeding frequency of 2 meals or 4 meals a day with the same amount of daily protein during 25 days has no effect on dietary protein utilization in growing female rats.
In the papers concerning the rules for proper nutrition we can find the recommendation for eating three meals a day. While treating obesity, feeding older persons and children, eating 4 to 5 meals a day seems to be more advantageous. Leciejewska  showed the existence of significantly higher thermogenic response and as a result worse energy utilization in people having two smaller meals a day as compared with one bigger, whereas Bellise et al. suggest  that there are no significant differences in daily energetic consumption while having meals at high or low frequency. In spite of this, there is a theoretical possibility that digesting and absorption of nutritional components consumed in the form of smaller but more frequent meals can affect the metabolic routes of their conversion in the post-meal period. Studies performed on laboratory animals showed, among others, a high rate of glycogen synthesis and lipogenesis de novo occurring after one big meal, whereas it did not occur following a few smaller meals . At the same time the possibility of improving the use of protein achieved by consuming a larger number of meals at a constant protein level in meals support further results of studies on animals, performed among others by Batterham and Bayley , Weijs et al. , Schiffelers et al.  and Bujko et al. . On the other hand, the studies performed by El-Khoury et al.  did not confirm any significantly advantageous impact of meal frequency on the utilization of proteins in humans.
The aim of this paper was to assess the interdependence between the dynamics of protein consumption and its utilization in the model experiments performed on laboratory rats of Wistar breed. This paper includes a hypothesis that the utilization of food proteins consumed during a day is influenced by the number and amount of meals.
Material and method
The experiment was performed on 36 young (5-6 week old) growing rat females of Wistar breed with mean body weight of 94 ± 6.8 g coming from the breed of Physiology and Nutrition Institute of Polish Academy of Science in Jabłonna near Warsaw. The animals were kept in animal quarters of the Faculty of Consumption and Human Feeding, SGGW (Warsaw Agricultural Academy) in single metal cages, in rooms under controlled conditions (temperature 22-23°C, air humidity 50-60%, daily light cycle 12/12 h) with permanent access to water. At the dark stage (8.00-20.00) of the daily cycle red light was used, enabling to perform necessary operations i.e. feeding and weighing the animals. The experiments included a standard diet: Labofeed B for rats (protein – 17.0%, energy – 12.15 MJ/kg) produced by the Feed Plant of Andrzej Morawski, ground before application in a metal (mill) grinder, then the feed was mixed with water at the proportion of 1:1. During the three days of initial stage aiming at getting animals used to the experimental feeding schedule, randomly selected half of animals (n=18) was fed ad libitum with two meals a day (group A; 9.00-10.00, 18.00-19.00). Remaining animals (group B; 9.00-9.30, 12.00-12.30, 15.00-15.30, 18.00-18.30) were fed ad libitum with 4 meals a day.
In the experimental period lasting 22 days for the first four days the feed consumption was monitored during the first meal, then mean (average) daily feed consumption was calculated for each animal. After this period (day 5) each rat from group A was assigned an animal from group B matching them by body weight. Animals in pairs (n=18) were given the same amount of feed divided into two meals (animals A) or four (animals B) animals. The dose of feed was defined based on mean daily consumption of a rat from group A, receiving feed two times a day. The body weight of those animals was checked everyday before the first feeding. During the first proper period the amount of feed every 4 days for each pair of animals was increased proportionally to body weight of the rat that received 2 meals a day (per 10% of growth the feed amount was increased by 8%). The feeding procedure was continued for next 5 days, then the animals on day 10, preceding the first 24-hour urine collection in metabolic cages were given glycine marked with isotope N15 (Sigma Aldrich Chemical Company, St. Louis, USA) at the amount of 0.5 mg/gram of feed in each meal. Application of marked glycine was continued till the next 24-hour urine collection (on day 22). The urine collection was carried out in metabolic cages made of plexi-glass (Techniplast, Italy, 320 cm3) every 10 days. The collected urine after acidification with 10% sulphuric acid solution  was frozen at the temperature of -22°C and stored for further chemical analyses. After 25 days of experiments animals were put to sleep to collect organs (heart, liver, kidneys, small intestine, soleus and gastrocnemius muscle), which after rinsing in physiologic salt solution and drying were weighed at accuracy of 0.0001 g. The rest of animal carcasses were frozen at the temperature of –22°C and stored for further analyses.
The total nitrogen content was determined by using Kjeldahl method with automatic analyzer type Kjeltec Auto 1002 ANALYZER manufactured by Foss Tecator company . Raw fat content was determined by using Soxhlet method. Dry matter weight and ash weight was determined with the method described in Official methods of analysis of The Association of Official Agricultural Chemists  in the laboratories of the Section of Dietetics and Functional Food, Dietetics Department of Warsaw Agricultural University (SGGW), Warsaw. The analysis of 15N content in rat urine was performed by using mass spectrophotometer ANCA GSL manufactured by an English company Europa PDZ in the Department of Dietetics, SGGW. Urea concentration in urine was determined by using a COBAS Integra appliance in the Central Laboratory of the Independent Central Public University Hospital In Warsaw with a set of reagents by Roche Diagnostics.
The studies granted permission from the III Local Ethics Commission at SGGW.
The results were analyzed by using the T student test for independent data (coupled in pairs) .
No statistically significant differences were observed at the body weight growth in rats receiving the same amount of food in two or four meals The average daily (24-hour) body growth amounted to 2.0 g/24h in animals receiving 2 meals a day, whereas 2.1 g/24h in rats receiving 4 meals. Statistical analysis did not show any significant differences in final body weight between animals receiving feed in two meals for 25 days (144±16.7 g) or in 4 meals (146±18.0 g). The average feed consumption per 100g of body weight amounted to 13.4 g/24h in animals fed twice a day and 13.2 g/24h in rats receiving 4 meals.
Data concerning the composition of body presents Table 1. The chemical composition of body was determined in animal carcasses obtained after removing examined internal organs (heart, liver, kidneys, small intestine, soleus and gastrocnemius muscle), the results were expressed in the percentage of fresh weight. The weight of those bodies was by about 8% lower than animal weight determined intra vitam.
Table 1. Chemical composition of rats´ carcasses.
|Group ||Dry mater [%] ||Protein [%] ||Fat [%] ||Ash [%]|
|A (2 meals) ||32.9?1.41||18.1?1.80||8.5?1.88||3.83?0.11|
|B (4 meals) ||32.5?1.74||17.9?1.78||8.0?2.13||3.92?0.06|
Statistical analysis did not show any significant differences in chemical composition of rat bodies being assessed: between those having two or four meals a day.
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