The Role of Nutrition in the Treatment of Sarcopenia in Old Patients: From Restoration of Mitochondrial Activity to Improvement of Muscle Performance, a Systematic Review

Sarcopenia is an age-related disease characterized by loss of muscle strength, mass and performance. Malnutrition contributes to sarcopenia pathogenesis. The aim of this systematic review is to analyze existing evidence on the efficacy of nutritional supplementation on muscle and mitochondrial health among sarcopenic or malnourished older adults. We included randomized controlled trials (RCTs) assessing the effect of branched-chain amino acid (BCAA), vitamin D and/or omega-3 polyunsaturated fatty acid (PUFA) on muscle mass, strength and performance and/or on mitochondrial activity and redox state in older sarcopenic and/or malnourished adults. The literature search was on MEDLINE, Embase and Cochrane Central, restricted to articles published in the last 10 years (2012–2022). Twelve RCTs with a total of 1337 subjects were included. BCAA with vitamin D significantly ameliorates appendicular muscle mass (4 RCTs), hand grip strength (4 RCTs), gait speed (3 RCTs), short physical performance battery (3 RCTs) or chair stand test (3 RCTs) among six out of nine RCTs. BCAA alone (2 RCTs) or PUFA (1 RCT) were not effective in improving muscle health. Mitochondrial function was significantly improved by the administration of BCAA alone (1 RCT) or in association with vitamin D (1 RCT). In conclusion, BCAA in association with vitamin D may be useful in the treatment of sarcopenia and boost mitochondrial bioenergetic and redox activity. PROSPERO CRD42022332288.


Introduction
Sarcopenia is a condition characterized by loss of muscle strength, mass and performance.Sarcopenia reduces independency, increases risk of falls and hospital admissions, reduces mobility and increases mortality.This disease is frequently associated with aging and plays a major role in the development of frailty syndrome [1].The worldwide prevalence of sarcopenia in people older than 60 years is estimated between 10% and 27%, the use of different diagnostic criteria [2] contributes to the under-diagnosis and under treatment of this disease.The ancient criteria essentially considered muscle mass reduction, whereas the more recent criteria take into account mainly the reduction of muscle strength and performance; different diagnostic criteria are summarized in Table 1.
with unhealthy aging at a clinical level and play a paramount role in the development of sarcopenia [15].
Amongst clinical factors associated with sarcopenia, malnutrition plays an important role.Malnutrition is a complex syndrome with multifactorial causes due to both a quantitative and qualitative reduction of dietary intake and an impairment of gut absorption, and it is frequent amongst older adults.This condition has been defined as an involuntary weight loss of 5% in less than 6 months or 10% in more than 6 months, or a BMI of less than 22, in patients over 70 years old, associated with reduced energy intake or acute injury or chronic-related disease [19].Several screening tools for the diagnosis of malnutrition have been proposed; in older persons, the more frequently used screening tools are the Mini Nutritional Assessment (MNA) or its short form (MNA-SF) [20,21] and the Nutritional Risk Screening Score (NRS) [22,23].These tools are user-friendly questionnaires.The three questionnaires focus on BMI, reduced food intake, weight loss and the presence of acute and severe illness.Within the MNA and MNA-SF, the presence of cognitive impairment or depression is considered as well as the patient's mobility.The full form of the MNA also takes into account dietary habits, self-perceived health, medication, living environment, skin wounds, and calf and arm circumference.The decreased intake/absorption of nutrients essential for muscle health, such as protein, amino acids, vitamins and minerals in malnourished subjects contributes to the development of sarcopenia [15,24].
The negative impact of reduced dietary intake of essential amino acid on muscle synthesis [25] in older age is enhanced by the reduced muscular protein synthesis during aging [14,26], this phenomenon has been named "anabolic resistance" of aging.The impairment in mRNA translation and in the activation of the mammalian target of rapamycin (mTOR) pathway, are amongst the underlying causes of this anabolic resistance.Higher protein intake can counteract the anabolic resistance [27].Indeed, branched-chain amino acids (BCAAs), particularly leucine, isoleucine and valine, have the capacity to activate the mTOR pathway [28], promoting mitochondrial biogenesis, and boosting the PGC 1 alpha pathway which increases oxidative resistance [14,15,29].Omega-3 PUFA also contributes to the activation of the mTOR pathway, leading to increased protein mitochondrial synthesis in muscle cells [30].Furthermore, omega-3 PUFA have an anti-inflammatory effect [31] and can decrease oxidative stress [32] thus counteracting the catabolic effect of inflammaging.
Vitamin D plays a role in maintaining muscle health and, in particular, it influences muscle cells differentiation, maturation and growth [33].Aging is associated with reduced expression of muscle vitamin D receptor and lower vitamin D level, and these factors contribute to the reduction of muscle mass [34].Furthermore, at the cellular level, some evidence shows that the active form of vitamin D, 1-25 dihydroxyvitamin D3 or calcitriol, increases mitochondrial biogenesis and function [35], and regulates the expression of genes involved in this process in muscles [36].
Given the role of malnutrition in the pathogenesis of sarcopenia, nutritional interventions targeting mitochondrial health and oxidative stress imbalance are emerging as possible strategies to treat this disease.
The aim of this systematic review is to analyze existing evidence on the efficacy of nutritional supplementation on muscle and mitochondrial health among sarcopenic and/or malnourished older adults.This review is divided into two parts: a clinical section focused on the role of nutritional supplementation on muscle strength, mass and performance and a biological section focused on the role of nutritional supplementation on mitochondrial health.

Study Design
We conducted a systematic review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [37], and Testing Methodological Guidance on the Conduct of Narrative Synthesis in Systematic Reviews [38], as well as Synthesis without meta-analysis reporting guidelines [39].The review protocol is published on the PROSPERO database (registration number CRD42022332288).

Inclusion and Exclusion Criteria
We included randomized controlled trials (RCTs) and meta-analyses which assessed the effect of nutritional supplementation the clinical parameters for the diagnosis of sarcopenia or the mitochondrial activity in older adults (65 years and older) affected by sarcopenia and/or malnutrition.The nutritional supplements analyzed were BCAA and/or vitamin D and/or omega-3 PUFA.
The research question was formulated by the following PICOS (participants, intervention, comparison, outcomes, study) strategy.

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Participants: Adults aged 65 years and older affected by sarcopenia and/or malnutrition clinically diagnosed.Sarcopenia was defined using recognized diagnostic criteria, such as the European Working Group on Sarcopenia in Older People (EWGSOP-1 [3] and EWGSOP-2 [4]), FNIH [5], International Working Group [6] or Asian working group on sarcopenia (ASIA) [7,40].The diagnosis of malnutrition or the presence of a malnutrition risk was defined according to a validated tool such as MNA, MNA-SF or NRS score [23].

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Intervention: BCAA and/or omega-3 PUFA and/or vitamin D supplementation, associated or not to physical exercise.

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Comparison: Standard clinical treatment, placebo, or physical exercise without nutritional supplementation.

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Outcomes: Measurement of the parameters of muscle mass and/or muscle strength and/or muscle performance or the mitochondrial activity and/or the oxidative stress.
We excluded trials whose participants were affected by sarcopenia due to the following diseases: kidney or hepatic failure, cancer, neuromuscular disease, AIDS, COVID-19, recent surgery or transplants, or severe neurological disorders.We also excluded clinical trials assessing the effect of any kind of protein, (soy, casein, whey proteins or unknown) alone without BCAA, vitamin D or omega-3 PUFA.

Study Selection
Two teams of reviewers worked, according to their expertise, on the clinical part (CC and GB) or on the biological part (FC and IB).The reviewers of each team worked independently on the studies retrieved by the search to evaluate the inclusion of each study; discrepancies between the two reviewers were solved through discussion and if necessary, by the third reviewer (PDA for both parts of the review).The Rayyan ® tool was used to detect duplicates and abstracts published before 2012 having escaped our filter, and to a systemized article selection process between the authors (available at Rayyan-Intelligent Systematic Review-Rayyan).The bibliography of included articles was screened to detect other eligible RCTs and subjected to the same process as the RCT detected by the searchstring strategy.

Data Extraction
From each study, we extracted the following data: authors, year of publication, location, number and characteristic of participants (mean age and gender), diagnosis at baseline (sarcopenia vs. malnutrition), type and duration of intervention, type of control group used, type of physical exercise, inclusion and exclusion criteria, and sample size and measured outcomes.If applicable, information on falls, length of hospital stay and mortality was also recorded.

Quality Assessment
The risk of bias was assessed by the RoB 2 tool according to Cochrane (https://me thods.cochrane.org/risk-bias-2(accessed on 14 July 2022)).As for the study selection, two reviewers independently evaluated the bias of each study; discrepancies between the reviewers were solved by discussion and consensus or by the third reviewer (PDA, for both parts of the review).

Data Synthesis and Analysis
The aim of our work was to write a systematic narrative review, without meta-analysis.To compare the different studies, we grouped them according to the type of intervention.Therefore, three subgroups were identified: 1. Vitamin D + BCAA or Whey protein; 2. BCAA alone; 3. Omega-3 PUFA.
For the biological part, with the first search strip, we retrieved 1542 abstracts, after searching the following databases: Medline (n = 776), Cochrane Central (n = 193) and Embase (n = 573).We removed 196 duplicates.A less restrictive search strategy was re-run and retrieved 652 more abstracts.We excluded 16 articles for violation of inclusion criteria and four articles were excluded due to unavailability of the full paper.Hence, two full-text articles were reviewed as previously described.Two articles were included in the biological part of this review and were also eligible for the clinical part [49,57,58,65].All the included articles were RCTs; no meta-analyses were included.
Figure 1 shows the study's flowcharts for both the clinical and biological part.
For the biological part, with the first search strip, we retrieved 1542 abstracts, after searching the following databases: Medline (n = 776), Cochrane Central (n = 193) and Embase (n = 573).We removed 196 duplicates.A less restrictive search strategy was re-run and retrieved 652 more abstracts.We excluded 16 articles for violation of inclusion criteria and four articles were excluded due to unavailability of the full paper.Hence, two fulltext articles were reviewed as previously described.Two articles were included in the biological part of this review and were also eligible for the clinical part [49,57,58,65].All the included articles were RCTs; no meta-analyses were included.
Figure 1 shows the study's flowcharts for both the clinical and biological part.

Study Characteristics
Amongst the 12 studies included in the clinical part of the review, seven evaluated nutritional supplementations with BCAA mixture in association with vitamin D [45,50,57,[60][61][62]64], one BCAA mixture alone [65], one leucine alone [58], two evaluated whey protein or proteins in association with vitamin D [59,63] and one omega-3 PUFA [47].All the characteristics of nutritional supplementation are specified in Table 1.Nine studies had a similar timing of intervention between 8 and 12 weeks; one was run for 30 days [64] and two considered a longer intervention period: six months [59] and one year [58].Amongst the included studies, four included a physical training program; in two, [60,61] physical exercise intervention including resistance training with gait and balance training was proposed both in the intervention and the control group.Two studies [45,63] were a four-arm trials comparing nutrition alone, exercise alone, nutrition plus exercise, versus no intervention [63] or health education [45]; exercise consists in resistance training [63] or in resistance and aerobic training [45].All characteristics of the studies are summarized in Table 2.

Study Characteristics
Amongst the 12 studies included in the clinical part of the review, seven evaluated nutritional supplementations with BCAA mixture in association with vitamin D [45,50,57,[60][61][62]64], one BCAA mixture alone [65], one leucine alone [58], two evaluated whey protein or proteins in association with vitamin D [59,63] and one omega-3 PUFA [47].All the characteristics of nutritional supplementation are specified in Table 1.Nine studies had a similar timing of intervention between 8 and 12 weeks; one was run for 30 days [64] and two considered a longer intervention period: six months [59] and one year [58].Amongst the included studies, four included a physical training program; in two [60,61], physical exercise intervention including resistance training with gait and balance training was proposed both in the intervention and the control group.Two studies [45,63] were a fourarm trials comparing nutrition alone, exercise alone, nutrition plus exercise, versus no intervention [63] or health education [45]; exercise consists in resistance training [63] or in resistance and aerobic training [45].All characteristics of the studies are summarized in Table 2.

Quality Assessment
Amongst the included studies, 16.6% raised some concerns or were evaluated at high risk of biases in the randomization process, 8.3% were at high risk from intended interventions, 16.6% were at high risk for missing outcome data, and 25% raised some concerns or were at high risk for reported result.Overall, 25% of the studies have high risk of bias and 8.3% raised some concerns (Figure 2).

Quality Assessment
Amongst the included studies, 16.6% raised some concerns or were evaluated at high risk of biases in the randomization process, 8.3% were at high risk from intended interventions, 16.6% were at high risk for missing outcome data, and 25% raised some concerns or were at high risk for reported result.Overall, 25% of the studies have high risk of bias and 8.3% raised some concerns (Figure 2).

Outcomes of the Included RCT
Five clinical studies out of nine testing vitamin D and whey protein or BCAA supplementation [59][60][61][62]64] found a significant improvement of muscle mass and/or on muscle strength in supplemented patients.Moreover, three studies [57,61,62] out of seven showed a significant effect of this intervention in the amelioration of physical performance; the studies that did not reach statistical significance showed a positive trend in the intervention group.Studies focusing on nutritional supplementation with vitamin D with whey proteins or BCAA administered with physical exercise [45,63] demonstrated a significant effect of the combined intervention on muscle mass, strength or performance.Regarding the biological findings, one study [65] assessing nutritional supplementation with a BCAA mixture showed a significant improvement of mitochondrial bioenergetics and redox activity.The second study showed a positive trend on mitochondrial activity with nutritional supplementation including a vitamin D, whey protein and BCAA mixture.Detailed results of intervention's studies are summarized in Table 3 for the clinical outcomes and Table 4 for the biological outcomes.

Outcomes of the Included RCT
Five clinical studies out of nine testing vitamin D and whey protein or BCAA supplementation [59][60][61][62]64] found a significant improvement of muscle mass and/or on muscle strength in supplemented patients.Moreover, three studies [57,61,62] out of seven showed a significant effect of this intervention in the amelioration of physical performance; the studies that did not reach statistical significance showed a positive trend in the intervention group.Studies focusing on nutritional supplementation with vitamin D with whey proteins or BCAA administered with physical exercise [45,63] demonstrated a significant effect of the combined intervention on muscle mass, strength or performance.Regarding the biological findings, one study [65] assessing nutritional supplementation with a BCAA mixture showed a significant improvement of mitochondrial bioenergetics and redox activity.The second study showed a positive trend on mitochondrial activity with nutritional supplementation including a vitamin D, whey protein and BCAA mixture.Detailed results of intervention's studies are summarized in Table 3 for the clinical outcomes and Table 4 for the biological outcomes.

Discussion
Sarcopenia is a frequent condition amongst older adults and malnutrition plays a causal role on its pathogenesis as well as mitochondrial dysfunction.The use of nutritional supplementation can be effective in improving mitochondrial function and in improving muscle health, thus treating sarcopenia; however, there is no clear recommendation on the type of nutritional supplements useful for treating this disease and ameliorating mitochondrial function.
Here, we suggest that nutritional supplementation with vitamin D combined with whey proteins or BCAA mixture can be effective in increasing muscle mass, muscle strength or performance, whereas supplementation with BCAA or omega-3 PUFA alone is not.All the studies on vitamin D used cholecalciferol in doses ranging between 100 and 1000 IU per day.Four out of nine studies on this combination demonstrated a significant gain in muscle mass [59][60][61][62] compared to placebo; however, no gain in muscle mass was observed if the control groups received nutritional counseling or a standard nutritional intervention.This result suggests that standard clinical treatment is sufficient to induce an improvement in muscle mass.
Nutritional supplementation with vitamin D combined with whey protein or BCAA may be effective in improving muscle strength [59][60][61]64], regardless of the addition of physical exercise.These findings are particularly relevant in geriatrics as, frequently, older patients are not willing to or are unable to follow an adequate physical activity program [66]; thus, we suggest to treat these patients with the combination of vitamin D and whey protein or BCAA, even without adding physical activity.
Three out of seven studies [57, 61,62] showed a significant improvement of the muscular performance thanks to the nutritional intervention with vitamin D and whey protein or BCAA.The four studies that did not reach statistically significant results showed a trend towards improvement of muscle performance [45,50,59,63].The included studies differed in the quantity of BCAA and whey proteins administered; the studies showing a significant improvement in muscle performance evaluated doses of total leucine higher than 3 g/day combined with 20 to 40 g of whey proteins/day, administered during the two main meals.Although there is no unique recommendation for the amount of BCAA required to trigger muscular protein synthesis in older patients [67], the literature suggests a dose-dependent correlation between BCAA supplements and muscular protein synthesis induction [68].It is noteworthy that the only study that did not find significant effects of BCAA on muscle mass, strength or performance [58] administrates leucin without other EAA or protein supplementation, suggesting that, although leucin is necessary to boost muscle health, it is not sufficient and must be administered with other EAA or with whey proteins in order to achieve a clinical benefit.According to Wolfe [69] and Wilkison [70], leucine alone might contribute to an enhanced anabolic response and therefore increase muscle mass, However, all the EAA are needed to induce muscular proteins synthesis [71].Hence, if only leucine is supplemented, the other needed EAA will be obtained from muscle protein breakdown, blunting the efficacy of leucine.
Although nutritional supplementation can boost muscular health even without physical exercise, adding aerobic or resistance training will significantly improve muscle mass, strength and performance [45,63].Physical inactivity increases anabolic resistance [14] and hence contributes to muscle waste and sarcopenia; moreover, physical exercise enhances the effect of BCAA and protein supplementation as muscle protein breakdown during exercise releases EAA that boost protein synthesis [69].A meta-analysis [72] published after the end of our research, including three RCTs, also included in our review [60][61][62], concludes that supplementation with whey protein, leucine and vitamin D, without physical exercise, increases muscle mass in sarcopenic patients; if nutritional supplementation is combined with physical activity, it also increases muscle strength and performances.Differently from Chang and Choo [72], we included in our systematic review not only supplementation with protein, leucine, and vitamin D in sarcopenic patients but also BCAA and omega-3 PUFA and widen the target population by also including malnourished patients.Despite these differences, our work supports their findings and contributes to generalization to a wider population.
Thus, we suggest adding appropriate and adapted training to nutritional supplementation whenever possible.
Only two studies evaluated the effect of nutritional supplements on mitochondrial activity: one study focused on BCAA alone [65], the other studied the effect of BCAA plus vitamin D [57].BCAA were proven to be effective in ameliorating mitochondrial bioenergetics and function even without the addition of vitamin D by Buondonno et al. [65].These authors showed an increase in the ability of mitochondria to produce ATP and an increased ability to reduce oxidative stress over two months.The authors also evaluated clinical outcomes on muscle mass, strength and performance, showing a positive trend on muscle performance; thus, they hypothesize that the improvement of mitochondrial activity via the mTOR signaling pathway and reduction of ROS may explain the improvement of muscle performance.
[57] evaluated the effect of nutritional supplementation with BCAA and vitamin D on mitochondrial activity showing that this treatment is effective in improving mitochondrial bioenergetics and function, and that this improvement might explain the amelioration of muscular performance; the authors demonstrated that patients with improved mitochondrial biogenesis had a significant improvement in the gait speed, whereas the others did not.
The results from these two studies suggest that nutritional supplementation may play a role in improving age-associated mitochondrial dysfunction, paving the way for further studies on other conditions associated with unhealthy aging, such as cognitive impairment.In this regard, Buondonno et al. [65] showed an improvement not only in muscular, but also in cognitive performance in old malnourished patients treated with BCAA, this improvement correlated with the increase in ATP production by mitochondria.
Even if some papers suggest that omega-3 PUFA may be beneficial in sarcopenic patients [30], we cannot draw any conclusions on their efficacy as only one study was eligible for our review [47].For instance, the results of this study were not significant; these authors tested omega-3 PUFA in addition to vitamin E that was also administered to the control group.After the conclusion of our search, an RCT tested the impact of supplementation with omega-3 PUFA (500 mg), leucine (2.5 g) and Lactobacillus paracasei PS23 in a population of sarcopenic patients [73].This study demonstrated a significant improvement in muscle mass, strength and performance in patients supplemented, compared to the placebo group.Although this study shows positive results, the researchers used a mixture of supplements; thus, it is not possible to conclude in favor of the supplementation with the sole omega-3 PUFA.
A major limitation for this review is the small sample size of the studies included and the presence of several biases in the study designs that are often not double blind and placebo controlled.Moreover, in this review, we could not evaluate the effect of vitamin D alone.Even though several authors showed that active vitamin D supplementation (calcitriol) significantly improves mitochondrial function, biogenesis and decreases oxidative stress [35,36,74], and, clinically, hypovitaminosis D induces muscle atrophy, clinical trials on vitamin D supplementation were not effective in proving its effectiveness on muscle mass, strength and function [75].
One of the strengths of our review is to focus on BCAA rather than on whey proteins; indeed, even if whey proteins contain BCAA (approximately 11%) [14], their quality, quantity and bioavailability might not be sufficient in order to activate muscle anabolism.
Another significant strength of our review is to include only studies on patients suffering from sarcopenia or malnutrition.This restriction allows us to reduce the heterogeneity between the studies as concerns the targeted population.
To the best of our knowledge, this review is the first to evaluate the impact of targeted nutritional supplementation on both biological and clinical outcomes and to shed light on their relationship.

Conclusions
Although there is still room for well-designed studies on the optimal treatment strategy for older malnourished patients, the results of this systematic review suggest that the treatment for these patients is vitamin D supplementation associated with either BCAA or whey proteins together with appropriate aerobic or anaerobic training.
All the studies included in this review used cholecalciferol in doses ranging between 100 and 1000 IU per day, and even at the lower doses [60], the supplementation of vitamin D coupled with BCAA and whey proteins is effective in ameliorating muscle health; hence, we recommend to give cholecalciferol 1000 IU daily in patients at increased risk of vitamin D deficiency according to the recent guidelines from the Society of Clinical and Economical Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Diseases (ESCEO) working group [76].
As regards the BCAA to be used, a mixture of amino acids including leucin, valine and isoleucine with a 2:1:1 ratio [65,77] or 4:1:1 ratio [45,60] works better than the sole leucine that might be used at doses equal to or higher than 3 g/day [69].If whey proteins are used, the intake needed to overcome anabolic resistance in older sarcopenic patients is at least 1.2 g/kg/day [78].These doses are above the internationally recommended dietary allowance of 0.8 g/kg/day [14,15,78] and can cause gastrointestinal problems or be contraindicated in case of kidney failure.These limitations in the use of whey proteins must be considered in the clinical prescription of nutritional supplementation in older patients.
Regarding the timing of protein intake, we suggest distributing proteins and or BCAA supplements over the three main meals to boost protein synthesis after each meal [14].
We also suggest using BCAA with or without vitamin D to boost mitochondrial activity and potentially ameliorate other chronic conditions associated with aging, such as cognitive impairment.
As concerns omega-3 PUFA, we cannot, based on the results of this review, recommend their use in sarcopenic older adults.

Figure 1 .
Figure 1.Study's flow charts.Panel (A) shows the flow chart of the clinical part and panel (B) shows the flow chart of the biological part.

Figure 1 .
Figure 1.Study's flow charts.Panel (A) shows the flow chart of the clinical part and panel (B) shows the flow chart of the biological part.

Table 2 .
Characteristics of the RCT included within the clinical and biological part.
Mean change in the intervention group: 8 ± 12, p 1.000 Mean change in the control group: 38 ± 10, p 0.003 Treatment*time interaction p 0.058 Dominant knee extension (Newton) Mean change in the intervention group: −2 ± 14, p 1.000 Mean change in the control group: 26 ± 21, p 1.

Table 3 .
Cont.Results showing an efficacy of the nutritional intervention as compared to placebo are highlighted in shadow.Efficacity of treatment are reported for all the studies RSMM: Relative Skeletal Muscle Mass; SMI: skeletal muscle index; AMS: Appendicular Muscle Mass; AMMI; Appendicular Muscle Mass Index; ALMI: Appendicular Lean Muscle Index; RSMI: relative muscle mass index; SPPB: Short Performance Physical Battery; EXNU: exercise in addition to nutrition; EX: exercise alone; NU: nutrition alone; HE: health education, CI: confidence interval.

Table 4 .
Biological outcomes.Results showing an efficacy of the nutritional intervention as compared to placebo are highlighted in shadow.Efficacity of the treatment is reported for all the studies.

Table 4 .
Cont.Results showing an efficacy of the nutritional intervention as compared to placebo are highlighted in shadow.Efficacity of treatment are reported for all the studies.