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1 From the Nutrition Unit, Estación Experimental del Zaidín, High Spanish Council for Scientific Research, Granada, Spain (IS, JD-A, CD-A and PN); the Institute of Nutrition and Food Technology, University of Granada, Spain (ML-F); and the Department of Pediatrics, San Cecilio University Hospital, Granada, Spain (AMH and GG)
2 Supported by a grant from the Spanish Ministry of Education and Science. 3 Reprints not available. Address correspondence to I Seiquer, Unit of Nutrition, Estación Experimental del Zaidín, High Spanish Council for Scientific Research, Camino del Jueves, 18100 Armilla, Granada, Spain. E-mail: iseiquer{at}eez.csic.es.
ABSTRACT
Background:Adolescents are nutritionally at risk because of their physiologic needs and dietary habits. Maillard reaction products (MRPs) are widely consumed by this population, mainly as a result of their high intake of fast food and snacks.
Objective:We compared the effects of diets with different MRP contents on dietary protein utilization in adolescent males aged 11–14 y. The brown diet (BD) was rich and the white diet (WD) was poor in MRP content (hydroxymethylfurfural: 3.87 and 0.94 mg/kg; fluorescence intensity: 21.04% and 7.31%, respectively).
Design:In a 2-period crossover trial, 18 healthy adolescent males were randomly assigned to 2 groups. The first group consumed the WD for 2 wk, observed a 40-d washout period, and then consumed the BD for 2 wk. The second group received the diets in the opposite order. Subjects collected urine and feces on the last 3 d of each dietary period. Fasting blood samples were collected after both periods.
Results:Compared with consumption of the WD, consumption of the BD resulted in 47% higher fecal nitrogen fecal excretion (P = 0.002), 12% lower apparent nitrogen absorption (P = 0.000), and a 6% lower nitrogen digestibility (P = 0.000). The apparent nitrogen retention and the utilization of ingested nitrogen did not differ significantly between the diets, although values after the BD tended to decrease. Serum biochemical variables related to nitrogen metabolism did not differ significantly.
Conclusions:The consumption of a diet rich in MRPs negatively affects protein digestibility. The possible effects of an excessive intake of MRPs during adolescence warrant attention, and long-term effects should be considered.
Key Words: Maillard reaction products • adolescent males • protein • nitrogen digestibility
INTRODUCTION
Adolescence constitutes a period of nutritional interest because of its greater dietary requirements for growth and development. Protein is essential during this phase of life because of its role in tissue accretion and maturation, and an understanding of the nutritional factors affecting protein utilization is crucial. Moreover, adolescence is a phase in which eating habits are promoted and consolidated and, thus, it is a potentially impressionable stage; it is also a time of growing independence, which includes greater opportunities to make decisions about what and when to eat (1). In recent decades, the dietary habits of adolescents have tended toward a greater consumption of fast food, which has increased from 2% of total energy in the late 1970s to 10% of total energy in the mid-1990s (2). The Maillard reaction (MR), also termed the nonenzymatic browning reaction, is usually developed in these kinds of foods. The MR commonly occurs during the thermal processing or storage of foods that are rich in proteins and reducing sugars, and it produces several compounds that contribute to the aroma, color, and flavor of cooked foods. Controlled browning is pursued in roasting, baking, frying, and some other food technology processes, and, thus, the MR products (MRPs) are widely consumed as a part of the human diet (3), especially during adolescence (4). However, the MR may cause degradation of nutritional protein quality (5, 6). This possible effect is due to the destruction of essential amino acids or a reduction in their availability (7, 8), decreases in protein digestibility as a result of structural changes (9), or even the inhibition of digestive enzyme activity (10, 11).
Despite the proven relation of the MR to protein quality, information on the effects of the consumption of diets rich in browning products in protein metabolism during adolescence is lacking. Adolescents often are a nutritionally vulnerable group, not only because their high physiologic needs but also because of their dietary habits. Therefore, the aim of this investigation was to study, in a sample of adolescent males, the utilization of protein in MRP-rich diets, as are usually consumed by this population, and to compare it with the corresponding values for diets low in MRPs.
SUBJECTS AND METHODS
Subjects
A total of 20 subjects were recruited among adolescent males aged 11–14 y and of medium-to-high socioeconomic status and education level who were living in the province of Granada (Spain). Their health was evaluated with the use of a medical questionnaire and physical examination. A nutritional survey of food habits and lifestyle was also conducted. Exclusion criteria included diseases or disorders that may modify the results of the variables under study, medications, eating disorders, and alcohol or tobacco use. Of the 20 subjects recruited, 18 completed the study; 1 subject dropped out because of a surgical intervention, and a second subject was found to be noncompliant.
The parents of the boys selected were invited to attend a meeting where the nature of the study was explained, and they provided written informed consent. This study was approved by the Ethics Committee of the San Cecilio University Hospital of Granada and was in accordance with the Helsinki Declaration of 1975, as revised in 1983.
Diets
To achieve diets that were balanced and adjusted to the nutritional requirements of this age group and to maintain the eating patterns of the subjects as much as possible, the experimental diets were designed on the basis of previous evaluation of this population’s habitual diet (12) and the recommended intakes for Spanish populations overall (13). Two 7-d menus containing the same daily servings of the different food groups were created. The 2 diets were of a similar energy and nutrient composition (Table 1). The foods included in the diets were basically the same, but the food processing was different whenever possible—eg, fried chicken rather than boiled chicken and fried potatoes rather than boiled potatoes.
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TABLE 1. Overall contribution of energy and nutrients of the designed diets and intakes during crossover dietary treatments with white and brown diets in adolescent males aged 11–14 y1
The white diet (WD) was free, as far as possible, of foods in which the MR develops during cooking practices (ie, frying, toasting, or roasting) or foods that naturally contain MRPs (eg, bread crust, chocolate, or coffee) The brown diet (BD) was rich, as far as possible, in processed foods with an evident development of browning, which therefore were rich in MRPs. This diet contained corn flakes, baked products, chocolates, fried foods, toasted foods, and breaded foods.
Food consumption was transformed into energy and nutrient intakes by using the computer program ALIMENTACIÓN Y SALUD (FOOD AND HEALTH; version 4.0), which was developed in the Institute of Nutrition of the University of Granada and is based on the Spanish Food Composition Tables database (14).
Lunch and dinner, the 2 main meals in the Spanish diet, were prepared by a local catering firm, under the strict control of the investigators (Table 2); they were distributed daily to the homes of the participants. The subjects and their parents were given specific instruction about the foods and the quantities of food to eat at breakfast and for the afternoon snack, which were prepared at home, for each one of the diets. For breakfast, the food composition of the WD was whole milk with sugar, crustless white bread and margarine, and fruit juice; the composition of the BD was whole milk with cocoa powder, breakfast cereal, and fruit juice. The afternoon snack in the WD was whole milk with sugar and a sandwich of crustless white bread and pâté or cheese and margarine; in the BD, it was whole milk with cocoa powder and pastries. The subjects were asked to avoid restaurant and carry-out food throughout the dietary treatments and to strictly comply with the recommendations.
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TABLE 2. A week’s lunch and dinner menus for the diets used for dietary treatments
To enable analysis of the protein content and of the MR indicators, the catering firm provided the investigators with the prepared foods from the complete menus; the breakfast and the afternoon snack were prepared in the laboratory according to the instructions given to the participants, the ingredients being purchased in a local market. Every day and for each diet, the edible portions of the foods from all meals were removed, weighed, and homogenized with a hand blender (Vital CM; Taurus, Barcelona, Spain); the inedible portions of foods (eg, chicken or fish bones or fruit skin) were not analyzed. Aliquots of each meal were mixed to obtain the 1-d sample, and aliquots of each day’s diet were mixed to obtain the WD and BD diet samples. Aliquots of meals and diets were lyophilized and stored at –20°C until they were analyzed.
Study design
A randomized, 2-period crossover design was used to compare the effects of diets that were rich and poor in MRPs on the utilization of dietary protein in adolescent males aged 11–14 y. The study lasted 70 d, and subjects were randomly assigned to 2 groups. The first group consumed the WD for 2 wk, observed a 40-d wash-out period, and then consumed the BD for the final 2 wk. The second group received the diets in the opposite order. The 7-d menu was consumed twice in each of the diets. The subjects consumed their usual diets during the wash-out period.
Compliance with dietary treatments was assessed throughout the dietary treatments by using daily record sheets on which participants recorded their food consumption. If the prepared meals were not entirely consumed, the subjects were asked to weigh and record all food that remained after a meal. They also weighed and recorded the foods consumed at breakfast and afternoon snack. These data were transformed into values of energy and nutrients (except protein) by using the FOOD AND HEALTH computer program.
In each 14-d dietary period, the subjects collected urine and feces on the last 3 d. The 24-h urine samples were collected in acidified containers, and the volume from each day was measured. The subjects were asked to report any problem with the collections, such as spillages or missed specimens. The presence of browning products was analyzed in the urine samples by measuring the absorbance at 420 nm (15) with a Spectronic-1201 spectrophotometer (Milton Roy, Rochester, NY); the absorbance values were related to the volume of the urine. Fecal samples were weighed, diluted with 6N HCl, and homogenized with a hand blender (Vital CM). Aliquots were frozen at –20°C for future analysis.
After a 12-h overnight fast at the end of each dietary treatment, blood was collected by venipuncture, left to clot for 30 min, and centrifuged at 1700 x g for 15 min at 4°C to obtain serum. Aliquots were frozen at –20°C for later analysis. Body weight and height were recorded at the beginning of the study and at the end of each diet period, and body mass index (BMI; in kg/m2) was calculated.
Measurement of MR indicators in the diets
Furosine
Furosine was measured by using HPLC according to the method and instrumental conditions described by Delgado et al (16) with some modifications. Briefly, 40 mg of the sample was hydrolyzed with 3 mL of 7.95 mol HCl/L at 110°C for 23 h. The hydrolysates were centrifuged at 14000 x g and 4°C for 10 min, and a volume of 0.5 mL was applied to a Sep-pak C18 cartridge (Millipore, Billerica, MA) and eluted with 3 mL of 3 mol HCl/L. The dried sample was dissolved in 1 mL water, acetonitrile, and formic acid (95:5:0.2). The mobile phase was 5 mmol sodium heptane sulfonate/L, which included 20% acetonitrile, 0.2% formic acid, and an Extrasyl-ODS2 analytic column (25 x 0.40-cm, 5-µm particle size; Tecknokroma, Barcelona, Spain). Furosine was quantified by the external standard method. The calibration curve was built from a stock solution (1.2 mg furosine/mL) in the range of 42.8 to 1.2 mg/L.
Hydroxymethylfurfural
HMF was measured by using HPLC according to the method of Rufian-Henares et al (17) and the same instrumental conditions. The sample (100 mg) was suspended in 1 mL 5% acetonitrile and clarified with 0.125 mL each of Carrez I [15% (wt/wt) potasium ferrocianide] and Carrez II [30% (wt/wt) zinc acetate] solutions. The resulting mixture was centrifuged at 14000 x g and 4°C for 10 min, and supernatant fluids were filtered for analysis of hydroxymethylfurfural content. A degassed mobile phase of water-acetonitrile (95:5) and the same analytic column described above was also used. Hydroxymethylfurfural was quantified by the external standard method within the range of 2 to 100 µmol/L. The HPLC system consisted of an MD-420 pump, an MD-465 autosampler, an MD-432 ultraviolet-visible detector, and DT-450/MT software (version 3.90) computing integrator connected to a PC (all: Kontron Instruments, Milan, Italy).
Fluorescence
Fluorescence associated with MRP was measured by using HPLC according to the method of Morales et al (18). Briefly, 100 mg of each sample was weighed and mixed with 1 mL 20% trichloroacetid acid. The solution was stirred and centrifuged at 14000 x g and 4°C for 10 min. Supernatants were filtered (0.45-µ acetate filters) and diluted to a concentration of 5 mg sample/mL to prevent quenching effects. The final solutions were measured at an excitation wavelength of 347 nm and an emission wavelength of 415 nm. The linearity of the fluorescence response was tested with a quinine sulfate solution of 1 µg/mL dissolved in 0.1 mol H2SO4/L. This solution was assigned 100% of fluorescence intensity (FI), and the results were expressed as the proportion of fluorescence in the quinine sulfate solution. A fluorescence spectrophotometer (SMF-25; Kontron Instruments) was used to measure fluorescence. Quartz glass cuvettes (QS-1.000 Suprasil; Hellma GmbH & Co, Müllheim, Germany) with light path of 1 cm were used.
Measurement of dietary nitrogen utilization
All analyses were performed in duplicate. Total nitrogen in foods, urine, and feces was analyzed by using a Kjeldahl procedure with mineralization (Block Digestor Selecta S-509; J.P. Selecta, Barcelona, Spain), distillation units (Büchi Laboratoriums Technik AG, Flawil, Switzerland), and titration units (Metrom AG, Herisau, Switzerland). Nitrogen was analyzed separately for all of the menu meals and the foods making up the breakfasts and afternoon snacks. Pools of feces, urine, and diet were used as an internal control to assess precision. The interassay CV was 0.64% in urine, 0.84% in feces, and 3.02% in the diet. Nitrogen intake over the 14-d period of dietary treatment was individually calculated by using the daily record sheets provided by the participants. The nitrogen values were converted to protein intake by multiplication by a factor of 6.25. Mean daily intake over the complete period was used to assess dietary nitrogen utilization, because it did not differ significantly from daily nitrogen intake over the last 3-d period. Using the data obtained for the nitrogen intake (I) and fecal excretion (F), the apparent absorption and the apparent fractional absorption or digestibility were calculated as A = (I – F) and %A/I = (A/Ix 100), respectively.
Most studies of nitrogen balance measure only urinary and fecal losses, but calculation of the nitrogen balance requires, in addition, the estimation of losses through skin, primarily in sweat (19). In that study, Rand et al reviewed dermal nitrogen losses in several reports of nitrogen balance studies and proposed data to be applied when such losses are not measured directly. Studies in preadolescent boys suggest that dermal or sweat nitrogen losses are 11.9 mg · kg–1 · d–1 (20), which coincided with the data of Rand et al (19) for adults if the study is conducted in an area with a hot climate. In the current study, therefore, nitrogen losses were corrected by a constant of 11.9 mg · kg–1 · d–1.
With the use of data obtained for urinary excretion (U) and the constant for estimated dermal losses (D), the apparent nitrogen retention was calculated as R = (A –U –D). In addition, the efficiencies of utilization of nitrogen that was absorbed [or biological value: %R/A = (R/Ax 100)] and ingested [%R/I = (R/Ix 100)] were ascertained. Uric acid, urea, creatinine, and total protein in serum were analyzed by using a Hitachi 917 autoanalyzer (Boehringer Mannheim, Mannheim, Germany). Serum protein fractions were measured by using capillary zone electrophoresis (Paragon CZE 2000; Beckman Instruments Inc, Fullerton, CA).
Statistical analysis
SPSS for WINDOWS software (version 13.0; SPSS Inc, Chicago, IL) was used for data entry and statistical analysis. The experimental data obtained after the crossover dietary treatments were analyzed by using the repeated-measures analysis of variance (ANOVA) to ascertain the dietary treatment effects and to clarify whether the order of presentation of the diets had an effect. There were no order effects and no treatment x order interaction for any dependent variables. When a significant effect between dietary treatments was found, post hoc comparison of means was made by using Bonferroni’s test. Differences were considered significant at P < 0.05.
Sample size was calculated with the assumption of a 10% change in nitrogen apparent absorption, with a power of 80% (ß = 0.20) of detecting such a difference at P = 0.05 ( = 0.05). On this basis, a sample size of 16 was required. MRP indicators in diets were statistically tested by 1-way ANOVA, and Tukey’s test was used to compare means that showed significant (P < 0.05) variation.
RESULTS
In general, the compliance of the subjects with the diet and the urine and feces collection was good, and they were sufficiently cooperative. The collaboration of the parents was fundamental, because balance studies are often problematic, especially in adolescents. The initial characteristics of the subjects are shown in Table 3. Mean (±SD) height and height increased 2.09 ± 0.32 kg and 2.13 ± 0.36 cm, respectively, during the experimental period, and there were with no significant differences between groups. The BMI did not change significantly during the study.
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TABLE 3. Baseline characteristics of the subjects1
During consumption of the BD, only the intake of fat differed significantly (P = 0.023) from that in the WD (Table 1). This fact may be related to the preference of the adolescent males for fried foods, which were present only in the BD. Nevertheless, no significant differences in energy intake were observed between the 2 diets. The intakes of protein and fiber also did not vary significantly between the 2 diets.
The analysis of the MR indicators showed that the furosine content did not differ significantly between the diets (6.99 ± 0.45 and 6.37 ± 0.15 mg/100 g in the WD and BD, respectively), but that of hydroxymethylfurfural and the percentage of FI were significantly higher in the BD than in the WD (hydroxymethylfurfural: 3.87 ± 0.03 and 0.94 ± 0.01 mg/kg; FI: 21.04 ± 0.42% and 7.31 ± 0.35%, respectively). Data are the mean (±SD) of 3 determinations. These results confirmed the greater presence of intermediate and advanced MRPs in the BD than in the WD.
Daily nitrogen intake (g/d) did not differ significantly in the 2 dietary treatments (Table 4). In contrast, fecal excretion of nitrogen increased with consumption of the BD, both as expressed in g/d and when related to body weight (mg · kg–1 · d–1). As a consequence, the apparent absorption and the digestibility of the nutrient were significantly lower when the subjects consumed the BD than the WD (Table 4 and Figure 1). Assuming that the obligatory (
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TABLE 4. Dietary nitrogen utilization in adolescent males aged 11–14 y during crossover dietary treatments with white and brown diets
FIGURE 1.. Mean (±SE) percentages of absorbed nitrogen (A)/ingested nitrogen (I), retained nitrogen (R)/A, and R/I for nitrogen balance in adolescent males aged 11–14 y after crossover dietary treatments with white (low in Maillard reaction products; ) and brown (rich in Maillard reaction products; ) diets. The subjects consumed the 2 diets for 14-d periods with an intervening 40-d washout period, and they collected urine and feces on the last 3 d of each dietary period. n = 18. Nitrogen retention was corrected by a constant of 11.9 mg · kg–1 · d–1 (estimated dermal losses). Different letters indicate a significant difference, P < 0.001 (repeated-measures ANOVA followed by Bonferroni test).
No significant differences in biochemical serum variables related to protein metabolism were found after consumption of the diets (data not shown). All of the measurements were within the normal range. The color index calculated in the urine (absorbance x volume of urine in mL) was significantly (P = 0.012) higher when the subjects consumed the BD (940 ± 104) than when they consumed the WD (606 ± 56).
DISCUSSION
As is usual among population of Western countries, the protein intake in the current study’s subjects was above the international recommendations for adolescent males of 0.97 g · kg–1 · d– (21), but it never exceeded the safe upper limit, which is defined as twice the reference nutrient intake (22). Moreover, in the past few years, some investigators questioned the optimum protein intake in adolescents and suggested that this amount may be higher than current recommendations (22, 23). On this basis, the protein content in the diets used in the current study was not modified with respect to the habitual intake of the subjects, which was similar to that found in epidemiologic studies of Spanish adolescents (24).
One of the methodologic constraints of nitrogen balance studies may be their limited duration. The UNU/World Hunger Programme recommends an adjustment period of 5 d (25). Urinary nitrogen excretion becomes stable 3–8 d after a change in diet, and no significant differences in nitrogen excretion have been found at 10–14 d (26). Therefore, we consider that the design of the current study was acceptable, although we accept that nitrogen retention data could be overestimated, because it is well known that balance studies tend to overestimate nitrogen intake and underestimate nitrogen losses. Nevertheless, the apparent digestibility of nitrogen during both dietary periods showed values similar to those of the few studies found in the bibliography concerning nitrogen balance in adolescents consuming mixed diets (27). Nitrogen digestibility values of 92.2%, which are similar to those observed in the current study for the WD, were described in adults fed mixed diets of very low protein content (90 mg · kg–1 · d–1; 28). When the protein is exclusively of vegetable origin, digestibility is markedly lower (29); it should be noted that most (58.5%) of the dietary protein in our assay had an animal origin, as is common in developed countries. This fact could contribute to the good protein digestibility of the experimental diets, despite the higher protein intake.
The higher fecal nitrogen excretion and the lower values of nitrogen absorption and digestibility found during consumption of the BD may be related to the greater MRP content of that diet. Studies of the adverse effects of MR development on the protein digestion of foods have been performed mainly in vitro and in animals. Changes in the physical state of the proteins subjected to heat treatment make them less soluble and perhaps less susceptible to the digestive enzymes (30). Several authors have shown that heat-induced changes to protein caused by MRs may prevent the action of digestive enzymes at their specific cleavage sites, thus reducing proteolysis and protein digestibility (10, 11). Moreover, certain MRPs may inhibit the activity of digestive enzymes such as carboxypeptidase A and aminopeptidase N (31). Hydroxymethylfurfural is an inhibitor of carboxypeptidase A (32), and its content in the BD was significantly higher than in the WD.
The MR causes serious reductions in the availability of several amino acids, mainly lysine (10, 33), that may become as much as 50% blocked (8). It has been observed in pigs that losses in protein nutritive value were due mainly to and proportional to the deterioration of lysine and, to a lesser extent, to the decreased digestibility of other essential amino acids (8). The MR even decreases the digestibility of unaltered nitrogen (7). Furosine (-N-(furoylmethyl)-L-lysine), which is related to the early stage of the MR, is an indicator of the formation and presence of Maillard products such as fructoselysine, lactuloselysine, and lysinoalanine (34) and of the losses in available lysine (35). The nonsignificant differences in furosine content between diets confirmed that commonly consumed foods always contain some of these early products, as was pointed out by Ersbersdobler (34). However, hydroxymethylfurfural and fluorescence are indexes of the presence of intermediate and advanced MRPs (17, 36), and the higher content of these products in the BD than in the WD indicated the progress of the MR in the BD. The measurement of fluorescence is well correlated with lysine damage, as was shown in studies of various food products such as breakfast cereals and pan-fried salmon (37). Moreover, the progress of the MR as a result of the thermal processing and storage produces a decrease in available lysine content and an increase in hydroxymethylfurfural (38). The presence of HMF has been measured in browning foods, such bread with crust (39), which are widely consumed in the BD but not allowed in the WD.
The amount of MRP markers is related to the cooking temperature of foods, and broiling, frying, and roasting result in the highest levels (40). These cooking methods also cause significantly more lysine damage than do milder heat treatments (37). Thus, cooking processes such roasting and frying, which are widely used in the preparation of the BD, could play a role in the reduced nutritional value of the protein consumed as part of the BD. In contrast, the processes used in the WD, such as boiling, produce lower amounts of MRPs (40). Reductions in protein digestibility have been observed in browning foods, such as the crust of a heated mincemeat loaf (30) or the bread crust (11), that are present in the BD.
The reduction in protein digestibility in the BD, although significant, was only 6% of that in the WD. The slightness of this variation could be explained by the facts that, in the BD, the damaged protein represented only a part of the total and that the diet was a varied diet composed of foods that in many cases were common to both dietary treatments. Moreover, given the high protein level in the BD, a weak although significant reduction in nitrogen digestibility did not seriously compromise the apparently absorbed nitrogen.
We observed no significant effect of the dietary treatment on apparent nitrogen retention or on the efficiency of nitrogen utilization, although decreases in the biological value and in net protein utilization as a consequence of MR development have been described (6, 41). The greater intensity of the color observed in the urine after this dietary treatment (measured by means of the absorbance at 420 nm) could be due to the increased excretion of MRPs. Metabolic transit studies performed mainly on rats with early MRPs, premelanoidins and melanoidins, indicated that they are partially absorbed by the intestines and excreted slightly modified or unmodified in the urine (42). Studies in human volunteers showed that the urinary excretion of pyrraline, a product formed in the final stage of the MR and present in roasted foods, bakery products, etc, is strongly dependent on its dietary intake (43). In contrast with Amadori products, 5% of which is recovered in the urine (42), dietary pyrroline is not metabolized within the body and is almost completely excreted in the urine (43). Thus, the color differences observed in the urine when subjects consumed the BD may be attributed to the presence of dietary advanced MRPs.
Urinary nitrogen was unchanged after consumption of either the BD or WD diet, and, therefore, the nitrogen retained by the adolescent did not differ significantly in the 2 diets (Table 4), although there was a trend toward lower values in the BD. In any case, nitrogen was retained in excess to meet maintenance and growth needs during adolescence (44). The digestive modifications observed in the current study after the consumption of the diets did not seem to have a significant effect on protein metabolism, and, accordingly, the plasma protein profile did not find differences between the groups. Nevertheless, the efficiency of utilization of ingested nitrogen during consumption of the BD was 11% less than that during consumption of the WD, although the differences were not significant (Figure 1).
To the best of our knowledge, this is the first study carried out in adolescents that focused on the effects of the consumption of MRP-rich diets on dietary nitrogen utilization. The results with respect to digestive and metabolic protein utilization did not involve any risk to the health of the subjects under our experimental conditions. However, given the negative effect of the BD on nitrogen digestibility, even when a balanced and varied diet is consumed, special interest should be taken in the long-term effects of dietary MRPs on nitrogen utilization. Moreover, it should be borne in mind that the food habits of adolescents are changing toward monotonous and unbalanced diets with a considerable MRP content, and an increasing proportion of total energy intake is obtained from fast food and snacks. In these conditions, the effects observed in the current study could be aggravated. In Spain, it is among this population where modifications of the Mediterranean diet are most visible (24). The current study, therefore, would enable the design of coherent intervention policies for this important and vulnerable sector of the population.
ACKNOWLEDGMENTS
We are very grateful to the participants for their role in the study. We also thank Luis Lara for statistical assistance.
MPN and IS designed and supervised the study, were responsible for the data collection and the interpretation of the results, and wrote the manuscript; ML-F collaborated in the elaboration and evaluation of the diets; AMH and GG helped design the protocol, participated in the selection of the subjects, collection of the data and biochemical analysis; JD-A performed the laboratory analysis of nitrogen; CD-A performed the Maillard reaction product marker analysis. None of the authors had any personal or financial conflict of interest.
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