**5. Discussion**

This study evaluated the effects of exercise and BCAAs on biomarkers of immunity, total albumin, and the cognitive profile of institutionalized older persons. The main findings were that ME showed more proemint result, particullary with BCAA in the improve cognitive profile and muscle strength-related albumin levels in plasma and diminish the frailty status. Moreover, exercise induced slight changes on the pro-inflammatory marker TNFα.

Albumin levels tend to decrease with age, and this effect seems to imply an increased risk of complications and higher rate of mortality, morbidity, and disabilities such as sarcopenia and frailty [56]. Despite the key participation of albumin on the pH balance and ionic homeostasis in blood, most of the free fatty acid (and some other lipids) transport in the bloodstream is also performed by serum albumin [57]. Not surprisingly, the agerelated impaired albuminemia and elevated serum anion gap are known to be associated with hypertension, low cardiorespiratory fitness, and decreased renal function, which are common morbidities of advanced aged people [58]. Therefore, interventions that aim to sustain (or even increase) albuminemia in older persons could represent an important strategy to mitigate the harmful effects of aging and its comorbidities. In this respect, some studies have already shown that BCAAs apparently increases albumin levels in older persons suffering from malnutrition [59].

Our results showed that the serum albumin levels were efficiently sustained or even augmented, in exercising participants (both ME and ME + BCAAs groups) during the first 16 weeks of intervention (phase 1). However, the withdrawal of BCAAs during the washout period (phase 2) quickly decreased those albumin levels, especially in the BCAAs group. The prominent effect of exercise on albumin levels was evident since its levels in both ME and ME + BCAAs groups were fully restored after the phase 3 period (T3 to T4 time-points), whereas only partial recoveries were observed in albumin levels in the BCAAs group at the same time-point. Low serum albumin levels were shown to be the most relevant biomarkers associated with poor physical strength in the older persons [60].

It is broadly accepted that the regular practice of exercise training imposes metabolic, endocrine/physiological, immune, and cognitive adaptations that, among many benefits, can increase skeletal muscle mass and strength, thus, circumventing the deleterious effects of sarcopenia in older persons [61].

The chronic exercise-mediated adjustments on insulin/glucagon balance, thyroid, and steroid hormones, such as testosterone, cortisol, and estrogens, can also be involved in the enhancement of hepatic and protein muscle metabolism (proteolysis, proteogenesis, and protein turnover), with clear consequences on the circulating amino acid levels (e.g., glutamine and alanine), blood pH and electrolyte balance (hydric/ionic homeostasis), and renal functions [62].

However, it was reported that the putative effect of amino acid/protein supplementation in older women could be masked by sufficient daily protein intake, as we attested in all institutionalized participants in this study [63]. Thus, the proper mechanism behind this effect still needs to be fully understood for this special population. In fact, to our knowledge, this is the first study to show the potential of physical exercise associated or not with BCAAs supplementation to maintain serum albumin levels in older persons living in RCH.

Contrarily to the albumin results, the monitored inflammatory markers (IL-10, TNFα, and MPO) did not show significant alterations over time. Apparently, we can putatively suggest, that the physical exercise intensities reached in the sessions, as well as the BCAAs supplementation effect compared to the daily protein intake in this population, were not sufficient to induce a significant impact on the inflammatory status in the participants in this study. Other interventions with older persons have been able to show a strong anti-inflammatory effect of exercise training, but it seems that these results were observed for intervention periods longer than 16 weeks [43,44].

Interestingly, even though an increase in the levels of the pro-inflammatory cytokine TNFα was observed in the ME + BCAAs group from T1 to T2 and T3, this finding was accompanied by a proportional increase of the anti-inflammatory cytokine IL-10, since the TNFα/IL-10 ratio was not different in this group over time. Moreover, at the end of the intervention, TNFα levels significantly decreased in this group. In accordance with the literature, IL-10 is a key anti-inflammatory cytokine that acts by inhibiting systemic inflammation mediated by TNFα [64].

Concomitantly, BCAAs alone did not induce alterations in both IL-10 and TNFα levels. These results differ slightly from what is observed in the literature regarding this type of intervention on inflammatory status [65]. Based on the literature, there is a close interaction between the inflammatory status and aging, and in this respect, it is widely accepted that older persons, especially sedentary people, present a chronic, systemic, sterile low-grade inflammation associated with aging, a phenomenon named inflammaging [66]. It is highlighted that inflammaging plays an important role in the loss of lean mass, which leads to sarcopenia and frailty, as well as increases the risk of the development of diseases and comorbidities, such as cognitive decline, atherosclerosis, insulin resistance, etc. [67].

Despite the fact that literature defines the ability to induce an anti-inflammatory change as a hallmark of physical exercise, in general, our results did not corroborate this fact. It is paramount to mention that some factors could putatively influence the lack of significant results in the inflammatory analysis. Firstly, the occurrence of inflammaging and pathophysiological disturbances in our participants could be crucial for the response magnitude observed during the interventions here. Second, the low level of physical activity of our participants before the interventions could mitigate the benefits that would be achieved with the physical exercise sessions and, consequently, limit physiological adaptation. These factors, associated with polypharmacy, a high rate of comorbidities, and the small sample size that finished the study, may determine the lack of significant effects observed.

There is a consensus in the literature that physical exercise sessions stimulate the release of cytokines, such as IL-6, IL-10, and TNFα, in response to contracting skeletal muscles, which are responsible not only for tissue restoration and energy metabolism, but also for the adjustment of the systemic inflammatory status [68].As appealing as these effects are, physical exercise training also improves human antioxidant defenses as observed in several studies which may also justify the use of exercise interventions to counteract the progression of oxidative-related diseases [69].

There are solid pieces of evidence that the loss of muscle strength and power in the lower limbs, which is characterized by a decline of up to 50% in overall muscle strength from the age of 30 to 80 years [52,53] is associated with an increased incidence of falls.

Particularly, physical exercise training improves body composition, muscle strength, metabolic parameters, bone health, and functionality as well as reduces the risk of mortality, chronic diseases, cognitive deterioration, falls, and depression [70]. Here, we observed that only the ME + BCAAs group presented an improved physical performance in the 5TSS test. Neither ME or BCAAs alone were sufficient to mediate improvements in lower body strength. Only the combination of exercise and supplementation did so. This result was achieved probably due to multiple factors, from physiological to cognitive positive effects that were not directly assessed by the applied methodology here. According to the literature, the 5TSS test is an important performance test that invokes physical skills and abilities that could have been particularly developed during phase 3 of this study. The phase 3 of our study included walking activities, steps, and balance exercises, which mimic the participants' regular daily life activities.

It is important to point out that strength exercise training has been proposed as one of the most effective methodologies, presenting best results in bringing back safety in per-forming the common tasks of daily life, focusing on the optimization of neuromuscular function for better benefits [71].

Multicomponent programs combine aerobic and strength exercises, including other physical skills, such as balance and flexibility [54], in order to optimize the functional capacity of frail older persons [72], as well as to maintain their independence to perform basic activities of daily living [73]. Concerning supplementation, it was reported that branched-chain amino acids, particularly L-leucine, showed significant results in inducing hypertrophy in older persons and improving their functional capacity [58,59].

Taking into account that cognitive impairment is one of the main factors that cause morbidity and high health costs worldwide [74], our results show that physical exercise training, in association or not with BCAAs, was able to maintain the cognitive scores of the participants and could have important practical applications. Considering the population enrolled here (pre-frail and frail octogenarians) and the trend for the natural decline of their cognitive functions, the maintenance of those cognitive scores by exercise is, per se, a remarkable achievement. The literature supports the positive effect of BCAAs in older persons, to improve their mood state [75], the perception of fatigue, and their performance in a mental task [76], which are abilities that were not evaluated here. Leucine is important since it activates the mammalian target of rapamycin complex 1 (mTORC1) and the downstream phosphorylation of p70S6 kinase and 4E (eIF4E)-binding protein 1 (4E-BP1) and related signaling pathways [77]. The aging muscle is less responsive to lower doses of amino acids when compared to the young muscle and may require higher quantities of protein to acutely stimulate equivalent muscle protein synthesis [78]. Nevertheless, the dose and duration of BCAAs proposed here did not affect the cognition scores in our participants.

#### *Study Limitation and Perspectives for Future Researchers*

The entire study was conducted with human octogenarians and, given the difficulty to control several influencing factors in this type of population, this study had the additional merit of causing a minimal impact on their daily routines at the residential care homes. In addition, our results here represent real-world data reflecting the reality at residential care homes. We screened participants with disabilities and comorbidities that, although expecting high rate of dropouts and low motivational issues, we could accomplish the proposed goals with a reasonable number of participants. The execution of a controlled study over 40 weeks with such a particular population also introduces other limitations. We sugges<sup>t</sup> that the use of other methods of exercise training, such as the use of playful activities (dance and music sessions) might elevate the adherence of this population to the program.
