Obese women on a low energy rice and bean diet : effects of leucine , arginine or glycine supplementation on protein turnover

This study examined if leucine, arginine or glycine supplementation in adult obese patients (body mass index of 33 ± 4 kg/m2) consuming a Brazilian low energy and protein diet (4.2 MJ/day and 0.6 g protein/ kg) affects protein and amino acid metabolism. After four weeks adaptation to this diet, each subject received supplements of these amino acids (equivalent to 0.2 g protein kg-1 day-1) in random order. On the seventh day of each amino acid supplementation, a single-dose 15N-glycine study was carried out. There were no significant differences in protein flux, synthesis or breakdown. The protein flux (grams of nitrogen, gN/9 h) was 55 ± 24 during the nonsupplemented diet intake and 39 ± 10, 44 ± 22 and 58 ± 35 during the leucine-, glycineand arginine-supplemented diet intake, respectively; protein synthesis (gN/9 h) was 57 ± 24, 36 ± 10, 41 ± 22 and 56 ± 36, respectively; protein breakdown (gN/9 h) was 51 ± 24, 34 ± 10, 32 ± 28 and 53 ± 35, respectively; kinetic balance (gN/9 h) was 3.2 ± 1.8, 4.1 ± 1.7, 3.4 ± 2.9 and 3.9 ± 1.6. There was no difference in amino acid profiles due to leucine, arginine or glycine supplementation. The present results suggest that 0.6 g/kg of dietary protein is enough to maintain protein turnover in obese women consuming a reduced energy diet and that leucine, arginine or glycine supplementation does not change kinetic balance or protein synthesis. Correspondence


Introduction
Obese subjects have increased protein turnover, whole-body protein synthesis and oxidation compared with same age lean adults (1).It has been suggested that protein balance in obese subjects is not maintained with a protein intake lower than 70 g/day when energy is restricted to 1100 kcal/day (4.5 MJ) (2).Garlick et al. (3) have also shown that when energy is restricted, the nitrogen balance decreases and is improved by high protein intake.
Supplementation with amino acids themselves has not been tested as a protein-sparing method during low energy diets in the treatment of obesity, but as a source of nitrogen one can expect it to be effective.Moreover, some amino acids have known pharmacological properties which could be of potential benefit for protein metabolism (4), and the effect of supplementation with these amino acids during low energy diets on obese persons has not been reported.
Two amino acids, arginine and leucine, are particularly recognized to have pharmacological in addition to nutritional properties.Arginine (L-2-amino-5-guanidinovaleric acid) is an aliphatic amino acid which has an important role in immune function and stimulates the release of growth hormone by the pituitary gland (5).Arginine is also a physiological precursor of nitric oxide (endothelium-derived relaxing factor) and this has been suggested as an explanation for its hypotensive effect on healthy subjects (6).Leucine (L-2-amino-4-methyl valeric acid) is a branched chain essential amino acid that has important roles in protein and glucose metabolism, neurotransmitter synthesis and lymphoid tissue metabolism (7).
This study was intended to test if supplementation with arginine and leucine improves protein and amino acid metabolism in obese patients consuming a low energy diet.To determine if any eventual changes in protein metabolism would be due to their pharmacological properties, we also studied a period of supplementation with glycine, an amino acid with no such effects.All supplementation periods were compared with nonsupplemented (control) ones.

Subjects
Seven obese women aged 36 ± 8 years were studied.Each subject underwent detailed clinical evaluation and blood tests (complete hematologic cell count, plasma glucose, creatinine and urea levels) and, apart from obesity, no other acute or chronic ill-nesses were detected.No subjects were pregnant or breastfeeding.After the initial evaluation, a dietary history was obtained and each subject was instructed to eat a local rice and bean diet (see below) and to maintain their usual level of activity.
This study was approved by the local Ethics Committee.Written informed consent was obtained from each subject after detailed information about the purposes and risks of this research.

Study design
Each volunteer received a low energy rice and bean diet (4.2 MJ) with a total protein intake of 0.6 g kg -1 day -1 throughout the 8-week study period.This diet, which has been used in previous similar research protocols, does not have any amino acid deficiency and reflects a typical Brazilian diet (8)(9)(10).
After an adaptation period of 4 weeks, the rice and bean diet was supplemented with leucine, arginine and glycine in random order (equivalent to 0.2 g protein kg -1 day -1 ) for 7 days each, so that each subject participated in four study periods.Between 7-day supplementation periods, the subjects received a nonsupplemented rice and bean diet for 2 days (washout period).A period of 7 days on a nonsupplemented diet was also studied (control).
On the 7th day of each period, a 9-h whole-body 15 N-glycine single-dose study was carried out (11) after a 12-h fast.Each subject was given six isoenergetic, isonitrogenous meals at 2-h intervals, which provided half the total usual daily energy, protein and supplemented leucine, arginine or glycine intake (i.e., 2.2 MJ, 0.3 g protein kg -1 day -1 from rice and beans, and an amino acid supplement equivalent to 0.2 g kg -1 day -1 ).Two hours after the first meal, the bladder was emptied for baseline urine enrichment determination and a dose of 15 Nglycine (200 mg per subject) was given (0 h).
All urine was collected until the 9th h of study for urea and ammonia 15 N enrichment determination.Two blood samples were taken for urea 15 N enrichment determination at 0 h and 9 h (12).

Protein turnover
Flux was calculated from the amount of isotope excreted in urine in the end product (urea and ammonia) over the 9-h study period.In addition, the amount of label retained in the urea pool at 9 h was used to adjust the result of flux based on the label excreted in urinary urea (13).Flux was calculated as: Q = d x Ex/ex, where Q is flux, d is the amount of isotope administered, Ex is the amount of end product excreted, and ex is the amount of isotope excreted as the end product over the study period.The end product average flux was taken as the harmonic mean of the estimates of flux based on the excretion of urea-N and ammonia-N, and was used in the calculation of protein synthesis and breakdown rates (12).Protein synthesis and degradation were estimated from: Q = Nint + D = Ex + S, where Nint is nitrogen intake, D is the equivalent of protein degradation, and S is the equivalent of protein synthesis.

Sample analysis
Urine was collected into containers with 5 ml concentrated HCl.Samples were stored at -20 o C until triplicate analysis.Aliquots were taken for duplicate determination of total nitrogen, ammonia-N, urea-N, ammonia-15 N, urea-15 N ( 14) and free amino acids (15).Total nitrogen was measured by the Kjeldahl method.Urea and ammonia were extracted sequentially by Conway diffusion and titration for mass spectrometry analysis by alkaline aeration.Isotope enrichment was measured with a mass spectrometer (Anca 20-20, Europa Scientific, Cheshire, England).Plasma amino acid was analyzed by high-performance liquid chromatography (Shimadzu Corporation, Tokyo, Japan) after precolumn fluorescence derivatization with ortho-phthaldialdehyde (15).

Statistical analysis
The repeated measures design was used for statistical analysis, with one-way withinsubjects ANOVA (for repeated measures or amino acid supplement).Thus, the repeated measure factor had four levels: no supplement, leucine supplement, glycine supplement and arginine supplement, in random order (16).Post hoc comparisons were made using the Tukey test.Fasted state was compared to fed state by the paired t-test.P values of 0.05 or less were considered significant.

Results
Anthropometric and biochemical data are shown in Table 1.Body mass index and arm fat index decreased significantly from day one to day 35 (adaptation period) and then remained constant up to the end of the study (Table 1).Blood biochemical data did not change significantly.Urinary urea excretion was higher on the first day of the study as compared to any other study period and urinary urea excretion remained constant from week 2 to 8.
Subjects 2 and 3 were excluded from the repeated statistical analysis because samples from one study period were lost for both subjects for technical reasons.There were no significant differences in whole-body protein turnover between the period of nonsupplemented rice and bean diet intake and any period of leucine, arginine or glycine supplementation (Table 2).There were also no significant differences in plasma or urinary amino acid profiles between fasted or fed state or between the nonsupplemented and different supplementation periods (Table 3).

Discussion
In this study, a protein intake of 0.6 g kg -1 day -1 was sufficient to maintain protein turnover in obese women on a reduced energy diet.During the experimental protocol, as expected, there was a drop in anthropometric measurements, but from around the fourth week on, after the adaptation period, there was anthropometric and biochemical stabilization.This may reflect the occurrence of energy expenditure reduction in response to the reduced energy intake, a phenomenon demonstrated by other authors (17).For this research, this stabilization represented, in fact, an advantage, as it eliminated the possibility of misinterpretation of results due to changes in body weight.
The rates of whole-body protein flux were measured using a single oral dose of 15 N-glycine (3,13).There is evidence that the method of 15 N-glycine infusion employed in whole-body protein turnover studies (i.e., constant infusion, repeated or single dose) does not affect the results (3,13).Besides, the use of a single dose permits the repetition of the study for many times, allowing the follow-up of time course changes in protein synthesis and breakdown (3).For wholebody protein turnover studies it is assumed that the nitrogen pool is homogenous and that the nitrogen exchange among different pools is constant under the experimental conditions used.However, the hypothesis of homogeneity and stability of the metabolic pool of body nitrogen is valid only under exceptional circumstances (12,18).For this reason, the fact that in the present investigation the same subject was studied under different conditions seems to be advantageous.Also, since this permitted the comparison of results with baseline and with every other period, any possible methodological problem would have been diluted in the overall experiment (19).Although this method implies an oversimplification of the complex reactions of protein kinetics in vivo, when it is applied under controlled experimental conditions it affords useful information concerning the dynamics of human protein metabolism (20).
Previous studies have shown that 0.6 g protein kg -1 day -1 is sufficient to keep the nitrogen equilibrium in healthy Brazilian persons on a rice and bean diet (9,10).It is possible that obese subjects have the same protein requirements, which remain unchanged during the low energy diet, as also proposed by others.Solini et al. (21) found no differences in leucine flux, oxidation or non-oxidative disposal between non-diabetic obese women and normal women.Other studies conducted on eutrophic subjects receiving 0.5 g protein kg -1 day -1 (10,22) found a kinetic balance close to that of the present study.Vazquez et al. (23) showed that an increase of the protein composition of isoen-ergetic weight reduction diets (2.5 KJ/day) from 50 to 70 g/day leads to no changes in nitrogen balance.
In the present study, whole-body nitrogen flux (around 39-58 mg nitrogen kg -1 h -1 ) was higher than that obtained for control subjects (around 17-46 mg nitrogen kg -1 h -1 ) (10,13) with a mean body mass index of 25 kg/m 2 .Protein synthesis and breakdown were also higher than values reported in the literature for normal body weight individuals (10,11,22) but similar to those reported for obese adolescents (24).One of these studies (10) was done using the same protocol/environmental conditions as the present study.It should also be pointed out that the variation in the data obtained here for obese subjects was similar to that observed in other studies of protein metabolism (1)(2)(3)20,21,24).
Leucine, arginine or glycine supplementation (0.2 g kg -1 day -1 ) did not improve kinetic balance or protein synthesis compared with the baseline diet period.It is possible that, as the subjects had no changes in protein metabolism induced by the low energy diet, any eventual effects of supplementation remained undetectable.
Urine and plasma amino acid profile did not show any remarkable differences, with the exception of high mean plasma values for phenylalanine, leucine and methionine when compared to literature values for eutrophic women (25).Plasma amino acid levels were not modified by supplementation.Kihlberg et al. (19) have described high plasma levels of phenylalanine and leucine and also of isoleucine, valine, lysine, tyrosine, proline and glutamic acid in obese women.These results suggest that obese women may have a particular amino acid profile, but this should be confirmed by further studies.
In conclusion, the present study does not support the use of leucine, arginine or glycine supplementation during low energy diets for obese subjects with the objective of decreasing protein breakdown rate and/or increasing protein synthesis rate.Obese subjects are able to keep a stable protein metabolism on a diet containing 0.6 g protein kg -1 day -1 and providing an energy intake of 4.2 MJ/day.Further trials with longer follow-up periods are needed to determine if nitrogen kinetic balance is maintained during very low energy treatment regimens (or even drug treatments).In addition, we con-firm that single-dose protein turnover studies are reliable, fast, inexpensive and easy to carry out in hospitalized patients.They can be used in short whole-body protein turnover studies in patients for whom long, continuous isotope infusion with gas and blood collection would be difficult or even impossible.

Table 1 .
J.S. Marchini et al.Clinical, anthropometric and biochemical data of the volunteers throughout the experimental protocol.
Data are reported as means ± SD, with the number of subjects given in parentheses.*P<0.01 compared to day 1 (ANOVA/Tukey test).Fat arm index = triceps skinfold/arm length; muscular circumference = arm circumference -p triceps skinfold; body mass index = weight/height.

Table 2 .
Kinetic data after a single dose of15N-glycine during the periods of no supplementation and supplementation with leucine, glycine or arginine (in random order).
Data are reported as means ± SD.Repeated ANOVA: a<b, a<c, a = d, b = c = d.gN = grams of nitrogen.

Table 3 .
Plasma and urinary amino acids before (fast) and after (fed) the kinetic study in obese subjects receiving a low energy rice and bean diet.