Effect of amino-acid intake on physical conditions and skin state: a randomized, double-blind, placebo-controlled, crossover trial (2024)

Abstract

The objective of this study is to elucidate the effect of a supplement enriched with l-leucine, l-arginine, and l-glutamine on body compositions/skin conditions. Healthy young women (n=29) were allocated to a group (n=14) receiving an amino-acid supplement (600mg l-leucine, 250mg l-arginine, and 300mg l-glutamine) and a placebo group (n=15) receiving a supplement not-containing the amino acids. The amino-acid supplement and placebo were given twice/day for 6 weeks. After a wash-out (2 months) from the 1st test, the amino-acid group received the placebo and the placebo group the amino-acid supplement. The body compositions/skin conditions were measured 4 times (day 1 and weeks 2, 4, and 6) in each test. Percentage-change of muscle mass in the amino-acid group increased up to 4 weeks (p=0.05) and was higher than that in the placebo group (p=0.09). Skin texture estimated by the image processing of neck skin replica tended to increase in the amino-acid group at 6 weeks compared with that at 0 week, though there was no significant intergroup difference. In conclusion, the young adult women having no fitness habit showed the significant increase of the muscle amount and improvement tendency of the skin texture by the continuous intake of the amino-acid supplement.

Introduction

In 2000 the World Health Organization (WHO) has defined the healthy life expectancy (HALE) at birth as the average number of years during which one can enjoy healthy life without any care, and clearly stated its significance in human life. Since then, many people in the world have come to recognize that expanding HALE is important together with expanding average life expectancy. Health risks/problems including osteoporosis, skin damage, perimenopausal symptoms, and presbyopia have increased with aging in any people. It has been, however, thought that we can prevent various diseases caused by aging by suppressing or delaying the decline of body functions and as the result extend both HALE and average life expectancy. This concept is called “anti-aging medicine” and we have thought that daily habits of eating, fitness, and sleep, and cosmetic cleaning are included as the important factors that are essential for anti-aging. Consciousness about health and anti-aging is high especially in middle-aged and elderly, while most women of broad generation tend to have common desires to become beautiful and highly interest in weight control. Thus, the number of population which habitually takes health supplements has increased to result in the expansion of a worldwide market of healthy foods whose main purpose is to attain the anti-aging, slim figure, health maintenance, beautiful skin, and the like. Mainstream of the past may need beauty cosmetics, but healthy supplements having effects from the inside of a body have attracted more attention recently. As a result, sales target of various beauty supplements includes all generations and the consumption of amino-acid supplements has been expanding in the supplement market worldwide; thus, the growth of the demand for such supplements is not temporally but continuously.⁽¹⁾

The objective of this study is to elucidate the effect of a supplement enriched with l-leucine, l-arginine, and l-glutamine on body compositions/skin conditions, because physiological significance of these amino acids is quite important as described below. Therefore, the supplement used in this study was enriched with the three amino acids in addition to some vitamins.

The signature of the physical aging appears as the change in body compositions including the decrease of muscle mass and the increase of fat amount. The decrease of muscle mass will cause the drop of the physical capacity and the increase of risks such as falling. Muscle synthesis responding to assimilation of food taken decreases with aging probably due to the reduction of susceptibility in protein synthesis. At least a part of the reduction of susceptibility will be ascribed to the decrease of physiological concentration of l-leucine, which is one of branched chain amino acids (BCAAs).⁽²⁾ A study on aged rats confirms that l-leucine stimulated muscle protein synthesis.⁽³⁾ Pasiakos et al.⁽⁴⁾ describe that muscle protein synthesis was accelerated with a supplement enriched with l-leucine in convalescing person who had exercised and presume that the intake of l-leucine after exercise is effective for the promotion of synthesis of body proteins. Indeed, l-leucine affects a signal pathway involved in metabolic control.⁽⁵⁾ Metabolic syndrome is a worldwide social problem at present and a possibility that intake of l-leucine is effective in the prevention of overweight and diabetes was indicated.

l-Arginine is an essential amino acid for infants/children and under certain conditions for adults. When one suffers from an injury and an infectious disease, sufficient supply of l-arginine is desirable, because this amino acid is important in certain biological systems and is known to have a function to stimulate immune reactions through activation of T-cells.⁽⁶⁾l-Arginine is metabolized in vivo to nitric oxide (NO) which has diverse physiological functions such as vasodilation, the improvement of exercise ability, the improvement of capacity of nutrition supplement to each organ, the suppression of accumulation of fatigue substances, and the improvement of antioxidative property.^(7,8) Increase of growth hormone was also observed in blood after intake of l-arginine supplement.⁽⁹⁾

l-Glutamine is an amino acid whose amount is the highest in human muscle and plasma accounting for approximately 60% of total free amino acids in a body⁽¹⁰⁾ and is the major source of energy in intestinal mucosal cells, fibroblasts, lymphocytes, and macrophages. Though this amino acid is not an essential amino acid, it is an indispensable ingredient for the normal growth of human culture cells.⁽¹¹⁾ This amino acid is involved in the DNA synthesis as a precursor of nucleic acids and nucleotides.⁽¹²⁾ In addition, l-glutamate is the precursor of l-arginine which generates NO as described above⁽¹³⁾ and glutathione, the major biological antioxidant.⁽¹⁴⁾l-Glutamate is supposed to have an anti-oxidation capacity and reported to resolve diarrhea and improve an intestinal barrier function after its oral administration in infants with diarrhea or malnutrition.⁽¹⁵⁾l-Glutamine additionally participates in the immune function⁽¹⁶⁾ and is supposed to elevate the ability of anti-oxidation, organ protection, and immunity, and to support the metabolism, and the synthesis and degradation of proteins.⁽¹⁷⁾

There are many studies on the effects of amino acids including l-leucine, l-arginine, and l-glutamine on health. Each amino acid possesses specific functions described above, while combination of these amino acids is presumed to give different functions from those observed in the single amino acid. There are, however, few in-depth reports on the functions obtained by combining amino acids though we can access reports on the functions observed in the mixture of BCAAs. In addition, subjects in those studies were person with specific disease, aged people, or athletes though there are a few reports on the healthy young subjects.^(18,19) Based on these situation and backgrounds, we performed a clinical trial using healthy young Japanese women who have no fitness habits to make clear the function(s) caused by an amino acid-containing supplement enriched with the three amino acids, i.e., l-leucine, l-arginine, and l-glutamine. The trial is a placebo-controlled, double-blind, crossover study in which the subjects ingested the supplement enriched with the amino acids for a prescribed period, and cosmetic and health effects were examined by skin tests and observations of body composition change, respectively.

Materials and Methods

Results

Table3 shows the change of the body weight and body compositions of the subjects in the AA and P groups. The weight tended to decrease from the starting point (0 week) in both of the AA and the P groups without significant intergroup and intragroup differences. No significant difference was observed in the amount of body fat and the value of % of body in the P group throughout the testing period, while those in the AA group tended to decrease up to 4 weeks at which p value was <0.1 (vs 0 week). The % change of muscle mass from the baseline increased in the AA group, but decreased in the P group; as a result the % change in the AA group was significantly higher than that in the P group at 4 weeks (Fig.1). Muscle mass was likely to increase by the amino acid supplementation. Two-way ANOVA showed no significant time effects, but a tendency of group effect was observed for the % change of muscle mass amount [p=0.06, F(1, 196)=7.8].

The skin moisture content, one of the indices to evaluate the skin conditions, exhibited a tendency of increase from the baseline in the neck of subjects in both groups of AA and P (Table4). The skin moisture content at the forearm significantly increased at 2 weeks in the AA group and 3 weeks in the P group (data not shown). In addition, in comparison with the starting time, significant increase of the skin moisture content at the neck was observed at 4 weeks in the AA group (p<0.05) and at 2 and 6 weeks in the P group (p<0.01, respectively). The TEWL tended to decrease with time in the skin of the forearm and the neck in both groups; the decrease of TEWL in the forearm skin was significant at 6 weeks in the P group (data not shown). Table4 demonstrates that the TEWL in the skin of the neck significantly decreased from the baseline at 4 and 6 weeks in the AA group (p<0.05 and p<0.01, respectively) and at 2, 4, and 6 weeks in the P group (p<0.01, p<0.05, and p<0.01, respectively). Neither skin moisture nor TEWL in the skin of the neck were significantly different between the AA group and the P group (Table4). Skin elasticity was measured with a cutometer. Indices of skin elasticity used for the analysis were R2 indicating viscoelasticity of the skin, R6 showing the degree of the slack, and R7 indicating the degree of skin tension. As a result, none of the parameters examined showed significant intergroup difference in the skin of the neck in the AA group and the P group (Table5).

Next, the skin color was examined using a colorimeter according to the SCE procedure (Table5). In the skin at the inner side of the forearm and the neck in either the AA group or P group, the brightness increased and the increase at 6 weeks was statistically significant in the P group (p<0.01). Brightness of the skin at the neck in the P group was higher than that in the AA group at 6weeks without significant difference (Table5). Redness of the skin color tended to increase in the P group, while yellowness of the skin decreased significantly in the AA group at 6 weeks (p=0.05). The slight skin redness in the AA group was slight, but significantly higher than that in the P group at 6 weeks.

A replica of the skin was picked followed by estimating skin texture by an image processing of the skin replica. Uniformity of the skin texture is estimated by the ratio of volume rate of the skin texture to the rate of whole skin volume. The higher the ratio became, the higher the uniform texture achieved.

The % change of the skin texture of the neck was observed to significantly increase in the AA group at 6 weeks (p=0.03), while the forearm skin texture did not change in both AA and P groups (Fig.2).

Discussion

In this study, we examined the changes in the body constitutions and the skin conditions in healthy adult women having no habitual exercise during and after taking either supplement not containing amino acids (P group) or that containing l-leucine, l-arginine, and l-glutamine (AA group) for 6 weeks. In the study, the body constitutions and the texture of skin were improved in the subjects belonging to the AA group though the change of weight was similar in both AA and P groups. In the AA group, body fat tended to decrease, while body muscle amount increased up to 4 weeks in comparison with the starting time. Thus, there observed a tendency of intergroup difference in the increase of the body muscle amount which was higher in the subjects in the AA group than those in the P group. Based on these results, we concluded that the continuous intake of the amino acid supplement containing l-leucine, l-arginine, and l-glutamine for at least 6 weeks tended to increased the muscle amount of the young adult women. Paddon-Jones et al.⁽²⁰⁾ report similar result in which the intake of essential amino acids stimulated muscle protein synthesis in the young and elderly people.

Hereafter we will discuss the respective effects of the three amino acids on the present results.

The followings have been elucidated as the role of l-leucine in several studies hitherto conducted: (1) l-leucine is an amino acid indispensable for protein synthesis ⁽²¹⁾ and a potent activator in the protein synthesis in white adipose fat-cells via a similar pathway,⁽²²⁾ (2) the carbon skeleton of l-leucine is utilized for the production of ATP, and (3) l-leucine is an activator of the mechanistic target of rapamycin (mTOR) whose signaling is important in vivo to control metabolism and the multiply, growth, and survival of cells. Based on these results, the effects of intake of l-leucine have been extensively studied in the various situations such as aging, muscle disorders, protein/energy deprivation, overweight, and genuine diabetes;^(23,24) overall these results indicating that l-leucine is most likely to affect both muscle mass and fat mass in the various physical situations. Pasiakos et al.⁽⁴⁾ performed a clinical study in which the following two groups were compared after physical exercise; one group took essential amino acid supplement and another group l-leucine-enriched essential amino acid supplement. The latter supplement elevated the plasma concentration of l-leucine followed by activation of the muscle cells and muscle protein synthesis during convalescence of fatigue in certain types of exercises requiring stamina.⁽⁴⁾ In our study, similar results were obtained though the enrolled subjects did not perform habitual exercise. Katsanos et al.⁽²⁾ report that the promotion of a muscle protein synthesis by ingestion of l-leucine was different between young and elderly person; in young adults the enhancement of muscle protein synthesis by l-leucine occurred regardless of its ingested amount, whereas in elderly adults high concentration of l-leucine was required for the enhancement. There is a report that the intake of l-leucine did not change the body protein amount in aging model mice and elderly people, though l-leucine stimulated body proteosynthesis.^(25,26) Our result found in the healthy young women taking 1,200mg of l-leucine per day in the AA group was well compatible with the above results. Thus, the present results observed in the young adult may not be accepted in elderly people.

There are several reports describing the effects of l-leucine on the body fat reduction,⁽²⁷⁾ the increase of energy expense in an obese model mouse,⁽²⁸⁾ and the increase of the adipose tissue by 25% without increasing food consumption in a young rat.⁽²⁹⁾ However, since a survey using a simplified-food frequency questionnaire implemented during the experimental period of this study confirms no significant change in the amount and contents of diets taken by the subjects (data not shown), the change of body compositions in this study was supposed not to attribute to the change of dietary amounts and contents. When l-leucine was orally taken as a supplement, no change was observed in the intake of the diet,⁽⁵⁾ though there is a report describing that direct injection of l-leucine into the brain caused the restriction of intake of food.⁽³⁰⁾ In conclusion, l-leucine was likely to increase the muscle amount and decrease the body fat without significant changes in the weight and dietary habits.

l-Glutamine has the following effects or rolls in vivo: (1) control of manifestation of many genes related to metabolism, signal transduction, cell defense, and repair,⁽³¹⁾ (2) activation of intracellular signaling pathways, (3) an energy source of cells of the immunity and digestive systems with rapid turnover, (4) a precursor of glutathione, an important anti-oxidant in vivo,⁽³²⁾ and (5) control and stimulation of the turnover of proteosynthesis. Furthermore, restraint of proteolysis is reported in culture cells of the mouse skeletal muscle.⁽³³⁾ Therefore, other than l-leucine, l-glutamine may be involved in at least a part of the increase of the muscle amount through proteosynthesis in the AA group. Many researchers have tried to verify the effect of l-glutamine from the viewpoints of medical nutrition^(17,34) and elucidated that this amino acid is important in maintaining functional status of the bowel and good physical conditions, and acts as an energy source of cells in the immune and digestive systems. Actually, oral administration of 30g of l-glutamine to obese and overweight adults for 14 days resulted in the decrease of the ratio of Firmicutes to Bacteroides, a good biomarker for obesity.⁽³⁵⁾ Though the amount of l-glutamine taken in this study was much smaller and 1,200mg/day, there was a possibility that the intake of l-glutamate changed the composition of intestinal flora followed by reduction of the body fat. A natural moisturizing factor (NMF) consisting of various components including amino acids holds the key for the moisture content of the skin by maintaining water in the corneocytes, and thereby moisturization of the stratum corneum (SC) of the epidermal keratinocyte is achieved. Thus, the decrease of NMF is accompanied by the decrease of moisture in the corneocytes, disturbance of the arrangement of the corneocytes, formation of the dried and flaking skin, and aggravation of skin conditions. Main components of NMF are amino acids (approximately 40%) formed by degradation of filaggrin (filament aggregating protein). In our study, TEWL (indicator of decrease of moisturization) tended to decrease by the intake of amino acids in the skin of the forearm and the neck in both AA and P groups. The decrease of TEWL indicated the improvement of the barrier function of the SC and skin conditions are closely related to gastrointestinal functions and status. Constipated state will increase corruption products in the gut which in turn aggravate skin functions and conditions.⁽³⁶⁾l-Glutamine will maintain the gut in desirable conditions and is likely to have a function to maintain the homeostasis of organs including the gut and the skin. These results and observations are consistent with the improvement of skin texture (smoothness) at the crista and sulcus cutis examined by dermoscopic images in our study. l-Glutamine by which the environment in the gut was arranged and nutritional absorption efficiency was elevated is potentially associated with the skin texture improvement. In addition, l-glutamine is converted to glutathione whose strong antioxidant property is probably contribute to the improvement of skin conditions. Contrary to our expectation, however, the improvement extent of the skin conditions including the moisture content, TEWL, and viscoelasticity was approximately the same in the AA and P groups. This resultmay be explained by the effect of multi vitamins as suggested by Boyera et al.⁽³⁷⁾

l-Arginine plays the central role in several biological systems including immune functions⁽⁶⁾ and the intake of l-arginine-enriched supplement enhanced the potency of self-antitumor immunity, prolonged survival time, and inhibited the growth of tumor in a cancer model mouse by inhibiting the number of myeloid-derived suppressor cells.⁽³⁸⁾l-Arginine is a substrate of both arginase and nitric oxide synthase (NOS) and is converted to urea and l-ornithine by arginase and to NO by NOS. l-Ornithine is a precursor of l-proline and important in the growth and restoration of cells. l-Proline biosynthesized from l-arginine via l-ornithine is the main constituent amino acid of collagen proteins. There is a possibility that collagen regeneration in the dermis is promoted by l-proline converted from ingested l-arginine, and thereby viscoelasticity of the skin improved. NO expands the blood vessel and improves blood stream.⁽⁷⁾ In addition, a study on the effects of intake of l-arginine supplement in rats shows a significant increase of NO level in blood, a decline of an oxidation stress, and a prolongation of an exercise time.⁽⁷⁾ Shan et al.⁽⁸⁾ demonstrate that l-arginine supplementation caused the promotion of the bioavailability of NO and the elevation of the anti-oxidative potency. The present study has confirmed the improvement of skin texture and skin viscoelasticity in the AA group. This observation can be explained by the follows: (1) the enhancement of NMF production in the epidermis and collagen regeneration in the dermis caused by the increase of blood stream by NO produced from l-arginine and the increase of supply of nutritious food matters, (2) the restraint of an oxidation stress of cells caused by anti-oxidative activity of l-arginine, and (3) the promotion of collagen regeneration by l-proline biotransformed from l-arginine.

The results described above indicate that the intake of the supplement containing l-leucine, l-glutamine, and l-arginine for 6 weeks changed the body constitutions such as the increase of the muscle mass and the decrease of the fat amount. The highest muscle amount was achieved at 4 weeks of the intake with a statistically significant difference in comparison with that of the P group, followed by a slight reduction at 6 weeks, though the reason(s) for this remain unclear at present. The improvement of skin texture was attained in the AA group. These findings were attributable to the additive effects produced by the combination of the three amino acids. Astaxanthin has high antioxidant and anti-inflammatory activities, and long-term astaxanthin supplementation suppresses the deterioration of wrinkles, moisture content, and interleukin-1α levels generated by environmental factors, such as UV and dryness, in a clinical study.⁽³⁹⁾ In addition, the oral administration of a dried whole egg powder of farmed soft-shelled turtles, Pelodiscus sinesis, containing various proteins and amino acids inhibits the formation of advanced glycation end-products in the serum and skin of diabetic rats.⁽⁴⁰⁾ Based on these reports, it is expected that the supplements used in this study can be combined with ingredients with high anti-glycation and anti-oxidant properties to develop more functional supplements to the skin.

Although this study provides the comprehensive and in-depth insight into the effect of the combination of l-leucine, l-glutamine, and l-arginine on the body components and skin conditions in younger women, several limitations have to be discussed. First, there are no placebo arms for the single amino acid, which enable to clearly demonstrate the role of the amino-acid combination. Second, since the sample size was small and only younger women were enrolled, generalizability of the data needs to be regarded with caution.

In conclusion this study shows that the consumption of the amino acid mixture affected the body compositions and the skin conditions of the healthy young women, and we can expect novel utilities of amino acid supplement based on these effects.

Acknowledgments

We would like to express our appreciation to the subjects who participated in this research. We are particularly grateful for the assistance in the measurement and data analyses provided by Yuka Arai, Yuko Kakita, Akina Koga and Nanase Terada in this study. We would like to also express our thanks to Seiji Kurita, B.Sc., and Hiroshi Matsui, M.Sc., (Groupwide Research and Development, Noevir Co., Ltd., Shiga, Japan) for the preparation of test samples.

Conflict of Interest

No potential conflicts of interest were disclosed.

References