Tolerability of glutamine supplementation in older adults: a double-blind placebo-controlled randomized clinical trial

In this double-blind placebo-controlled randomized investigation, we assessed the tolerability of glutamine in older adults recruited from three daycare centers. The relevance of studying glutamine supplementation in elderly patients lies in its potential to provide a well-tolerated intervention. Glutamine, a crucial amino acid, plays a vital role in various physiological processes, including immune function and protein synthesis. Understanding its impact on older adults is essential, given the potential implications for their health and well-being. Participants received a daily dose of 12.4 g of oral effervescent glutamine (EGln group) or maltodextrin (placebo group) for 60 days. Fifteen patients from each group completed the study. The mean ages were 77.0±9.1 and 79.0±6.9 years for the EGln and placebo groups, respectively. We evaluated body mass index, aminogram, hemogram, plasma levels of glucose, prealbumin, albumin, urea, creatinine, uric acid, C-reactive protein, vitamin D, calcium, sodium, potassium, and the plasma activities of aspartate aminotransferase and alanine aminotransferase. Notably, we quantified a broad array of inflammatory markers and growth factors providing a holistic understanding of the potential effects of glutamine supplementation. The results demonstrated that oral glutamine did not induce significant changes in any evaluated parameters, and no adverse effects were reported. This finding suggested that the dosage of glutamine used in this study was well-tolerated and safe. This information contributes to the broader understanding of glutamine supplementation, emphasizing its safety and supporting its potential as a viable intervention for maintaining health in aging individuals.


Introduction
Aging is characterized by a gradual decline in the ability to adapt to the environment, a reduction in social standing, diminished functional capacity and independence, increased susceptibility to infections, a higher prevalence of chronic diseases, and an elevated risk of morbidity and mortality (1,2).Additionally, normal aging is marked by a low-grade chronic systemic inflammation, evident through elevated blood levels of inflammatory biomarkers (3,4).This inflammatory state is intricately linked to conditions such as insulin resistance, cardiovascular diseases, type 2 diabetes, and neurodegenerative diseases (5).
As the global population of older individuals continues to grow, it is crucial to develop strategies that can delay, alleviate, or even reverse the processes associated with senescence, senility, and frailty.
Despite the potential beneficial effects of glutamine, including activating the immune response, stimulating protein anabolism, protecting the intestinal barrier function, promoting an anti-inflammatory response (14)(15)(16)(17)(18)(19)(20)(21), and providing protection against oxidative stress (21), there is a limited number of studies involving glutamine supplementation in older adults (18,19,22).Additionally, there is a lack of investigations into the tolerability of this amino acid among the older adult population.Our hypothesis was that glutamine is safe for older adults, as previously established for younger patients (15,(23)(24)(25)(26).
To test this hypothesis, we conducted a double-blind placebo-controlled randomized study comparing older adults receiving oral effervescent glutamine (EGln) or maltodextrin (placebo) for 60 days.

Ethical approval
The protocol received approval from the State University of Maringá Standing Committee on Human Subject Research Ethics (COPEP 1.808.919).

Participants
We recruited forty-four individuals from three daycare centers registered with the Council for the Rights of Older Adults in Maringá, Brazil.
The eligibility criteria required participants to be: 1) aged 460 years and 2) willing to participate in the study.The geriatric doctor on our team (T.C.M.N.) made the final decision to include each patient, following an evaluation of their potential for treatment engagement, medical history, and lab test results.
Thirty-four individuals or their legal representatives completed, signed, and dated the informed consent form.Before initiating treatment, all patients underwent a clinical evaluation.

Patient randomization
To minimize potential biases in result interpretation and provide reliable evidence about the effectiveness of the medical intervention, a double-blind placebo-controlled randomized clinical trial was implemented.This ensured that both participants and researchers involved in the study were unaware of who was receiving EGln and who was receiving the placebo.
The enrolled participants were randomly assigned to two groups, each comprising 17 volunteers.This randomization process was employed to ensure that any observed differences between the groups were not influenced by pre-existing factors but rather resulted from the studied intervention.
The control group received a placebo substance (maltodextrin), and this group was then compared to the group receiving EGln.Both substances were packaged in sachets (12.4 g).Prior to ingestion, EGln and maltodextrin were dissolved in 200 mL of water, resulting in a concentration of 62 mg/mL.EGln and maltodextrin were consumed daily for 60 days.On Fridays, each patient received two sachets for use over the weekend.Maltodextrin was selected as the control substance due to its low cost, easy preparation, high solubility in water, and its prior utilization in our previous clinical study (15).
Body mass index (BMI) was evaluated before and after 60 days of EGln or maltodextrine supplementation.

Clinical evaluation and characterization of the participants
A questionnaire was administered to collect information on age, gender, race, medical history, educational level, marital status, therapeutic profile, and lifestyle.
To assess physical activity levels in adults the International Physical Activity Questionnaire (IPAQ) was utilized (27).The IPAQ is structured to provide a comprehensive overview of a person's physical activity patterns across various domains of daily life, including work, transportation, household chores, and leisure-time activities.The key components measured by the IPAQ include intensity, duration, and frequency of physical activity.
The Charlson Comorbidity Index is a scoring system that provides a standardized and quantitative measure of comorbidities, allowing healthcare professionals to assess the impact of these conditions on patient prognosis (28).
To evaluate how well older individuals can perform basic everyday activities on their own (bathing, dressing, moving in and out of bed or a chair, control of bowel and bladder function, using the toilet, and ability to eat independently) the Katz Index of Independence in Activities of Daily Living was used (29).This index helps us understand both the physical and mental aspects of their well-being.
The Lawton & Brody Scale (30) includes the selfmaintaining activities of daily living (SADL) and instrumental activities of daily living (IADL) subscales.SADL refer to basic self-care and physical well-being (bathing, dressing, grooming, and control over bowel and bladder).IADL involves more complex tasks that are crucial for independent living in the community (cooking, shopping, managing finances, using transportation, and performing household chores, etc.).Therefore, IADLs reflect a person's ability to function independently in a broader social context.These activities are important for maintaining a person's quality of life and ability to live in a community setting.
The International Clinical Practice Guidelines for Identification and Management of Physical Frailty provides a comprehensive approach to identify and manage physical frailty, emphasizing personalized and multidimensional strategies encompassing physical, nutritional, psychological, and social aspects (31).These strategies include questionnaires, physical performance tests, and other measures.Geriatricians play a crucial role in assessing an individual's overall health and medical history and conducting physical examinations to identify signs of frailty (weakness, slow walking speed, unintentional weight loss, low physical activity, and fatigue).

Preparation of EGln
Each sachet contained 12.5 g of glutamine (Ajinomoto North America, USA), combined with 3.90 g of sodium bicarbonate as the effervescent base (Arbros Industria Pharma e Alimentícia Ltda., Brazil), as well as 1.30 g citric acid and 2.60 g of tartaric acid (SM Pharmaceutical Enterprises, Brazil), which release carbon dioxide when dissolved in water to initiate effervescence (32).

Blood collection
Blood was collected after an overnight fast.Immediately after blood collection, a portion of fresh blood was used to measure glycated hemoglobin A1c and a complete blood count.Another portion of fresh blood was transferred to a tube containing sodium fluoride/ EDTA, which was immediately centrifuged (2000 g for 10 min at room temperature), and the plasma was separated for measurements of amino acid, biochemical parameters, and cytokines.

Biochemical parameters
Blood levels of glycated hemoglobin A1c, plasma concentrations of glucose, prealbumin, albumin, urea, creatinine, uric acid, C-reactive protein, vitamin D, calcium, sodium, potassium, and plasma activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined by lab tests using conventional kits according to the manufacturer's recommendations.

Statistical analysis
Analysis of variance (ANOVA) and the Tukey test for multiple comparisons were used to assess the differences in results within each group (EGln group or placebo group) on days 0, 30, and 60.Additionally, an unpaired Student's t-test was used to compare results between the EGln and placebo groups on the aforementioned days.The statistical analyses was performed using Statistica TM 8.0 software (StatSoft, Germany; https://www.statistica.com).Results are reported as means±SD.A significance level of Po0.05 was considered statistically significant.

Results
The mean ± SD age for the EGln group was 77.0 ± 9.1 years, ranging from 64 to 91 years, while for the placebo group, it was 79.0±6.9 years, ranging from 61 to 88 years.Each group had eleven women and four men.In the EGln group, there was one married, three divorced, and eleven widowed individuals, while in the placebo group, there was one married, one divorced, two single, and eleven widowed individuals.The level of education in both groups ranged from 0 to 8 years, with a mean±SD of 2.7 ± 2.7 years for the EGln group and 1.9 ± 2.3 years for the placebo group.
As conveyed by the geriatric specialist on our team (T.C.M.N.), all participants were of Caucasian ethnicity.In the EGln group, there was 1 smoker, 10 hypertensive individuals, and 4 diabetics while the placebo group had 13 hypertensive subjects and 4 diabetics.The average number of medications used by patients in the EGln group was 4.3 (ranging from one to eleven medications), whereas in the placebo group it was 5.7 (ranging from zero to seventeen medications).
Based on the IPAQ, all volunteers from both the EGln and placebo groups exhibited low levels of physical activity.
The Charlson Comorbidity Index estimated a lifespan of ten years for both groups, ranging from 2 to 77% for the ELGn group and 0 to 77% for the placebo group.
According to the Katz Index of Independence in Activities of Daily Living, nine individuals in the EGln group were classified as independent, four as partially dependent, and two as dependent.In the placebo group, nine were independent, six were partially dependent, and none were completely dependent.
The Lawton & Brody Scale revealed that two individuals in the EGln group were independent, seven partially dependent, and six dependent.In the placebo group, three were independent, six were partially dependent, and six were dependent.
Using the International Clinical Practice Guidelines for Identification and Management of Physical Frailty, we identified nine pre-frailty and six frailty cases in the EGln group and ten pre-frailty and five frailty cases in the placebo group.
During the treatment period, one patient from the placebo group passed away and three dropped out (one from placebo group and two from EGln group), while fifteen individuals each from the EGln group (n=15) and the placebo group (n=15) successfully completed the clinical trial.Furthermore, no participant reported any discomfort (such as nausea, dyspepsia, or other undesirable effects) during glutamine supplementation.
The BMI values before and after supplementation with EGln were 29.1±5.3 and 29.3±5.5, respectively.Similarly, BMI values before and after supplementation with maltodextrin (placebo) were 30.3 ± 5.3 and 30.1 ± 5.1, respectively.These results indicated that EGln and maltodextrin did not induce any significant change in BMI.
With the exception of threonine, the plasma levels of various amino acids (argininosuccinic acid, aspartic acid, glutamic acid, alanine, arginine, citrulline, glycine, histidine, leucine, isoleucine, methionine, ornithine, phenylalanine, proline, asparagine, serine, tyrosine, tryptophan, and valine) did not show statistically significant differences between the EGln and placebo groups on day 0 (before supplementation with EGln or maltodextrin) and on day 60 (after supplementation with EGln or maltodextrin) (Table 1).
Except for hematocrit, the blood parameters hemoglobin, MCHC, leukocytes, platelets, segmented cells, lymphocytes, monocytes, eosinophils, and basophils all exhibited values within the normal range before and after 60 days of supplementation with either EGln or maltodextrin (Table 2).
Fasting glycemia and glycated hemoglobin A1c values before and after 60 days of supplementation with EGln or maltodextrin indicated prediabetes, but no significant differences were observed between the EGln and placebo groups.Additionally, the plasma levels of prealbumin, albumin, urea, creatinine, uric acid, PCR, vitamin D, calcium, sodium, potassium levels, and plasma activities of AST and ALT all exhibited normal range and similar values on days 0, 30, and 60 in both the EGln and placebo groups (Table 4).

Discussion
There are few studies available on the plasma aminogram in older adults (33)(34)(35)(36).In this study, we observed no significant changes in aminoacidemia after 60 days of EGln supplementation.Additionally, glutamic acid, the primary metabolite of glutamine, remained unchanged after 60 days of EGln supplementation.
Hematological analysis plays a crucial role in the toxicological evaluation of a substance, offering valuable insights into its potential adverse effects on blood and its components (37).In the context of this study, EGln treatment did not adversely impact hematological parameters.The exclusion of volunteers with hemoglobin levels below 12 mg/dL, leukocytes counts above 15,000/mL or below 1,500/mL, and platelet counts below 100,000/mL contributed to the maintenance of normal range values for all hematological parameters following EGln supplementation.
Despite the well-known anti-inflammatory properties of glutamine (20,21,38), we generally did not observe significant changes in the plasma levels of inflammatory biomarkers after 60 days of glutamine supplementation.The only exceptions were the reductions in plasma levels of ITAC and MIF in the placebo group from day 0 to day 60, but these changes were not clinically significant.The exclusion of volunteers with C-reactive protein levels above 5 mg/dL and those with inflammatory diseases reduced the likelihood of detecting an anti-inflammatory effect of glutamine.
Consistent with results reported for young individuals (17-19,38-40), we hypothesized that glutamine supplementation would be well-tolerated in older adult subjects.This hypothesis was confirmed, as we observed no changes in BMI, plasma levels of creatinine (indicative of renal toxicity), AST, ALT (indicative of liver toxicity), and urea (reflective of protein catabolism).
To the best of our knowledge, there exists only one comparable clinical controlled, double blind, crossover study investigating the tolerability of glutamine in older featured a cross-over study with supplementation periods limited to 14 days, with a five-day washout period between treatments.The primary difference, however, lies in the daily dose of glutamine: 0.15 mg/kg Â day in our study compared to 0.50 mg/kg Â day in the study by Galera et al. (23).The approximately three times higher dosage in the latter study may explain why our study indicated the safety of glutamine, whereas in their study increases in serum urea nitrogen and creatinine and decreases in estimated glomerular filtration rate were observed.To comprehensively understand the implications for clinical practice and public health measures, further studies on the tolerability of glutamine in older adults are imperative for several compelling reasons.First and foremost, the current body of research is notably limited, with only one comparable clinical, controlled, double-blind study conducted on glutamine tolerability in this population (23).Additional studies are needed to validate and build upon the insights gleaned from the existing sparse research.Secondly, the diversity of older adults, Data are reported as means±SD (n=15/group).MRCV: Mean red cell volume.*Po0.05,ANOVA with Tukey test was used for multiple comparisons to assess the differences in results within each group on days 0, 30, and 60.
Table 3. Plasma cytokine levels (pg/mL) in older adults before (day 0) and after (days 30 and 60) supplementation with effervescent glutamine (EGln) or placebo (PL) at a daily dosage of 12.4 g.A critical aspect that merits exploration is the determination of optimal and safe dosages as well as duration of glutamine supplementation for older adults.This information is vital to make recommendations and ensure that older individuals can maximize the benefits with minimal risks.Moreover, diversifying the scope of research to include various control substances and placebos can enhance our comprehension of glutamine's effects.Additionally, investigating the long-term effects of glutamine supplementation is essential to assess its safety and tolerability among older adults over time.Expanding the examination to encompass a broader array of biomarkers and health outcomes will provide a more comprehensive picture of the multifaceted effects of glutamine supplementation.This includes exploring potential benefits related to muscle health and immune function, contributing to a more holistic understanding of the supplement's impact.
Considering that older adults often undergo concurrent medication regimens, it is imperative to investigate potential interactions between glutamine ingestion and commonly prescribed medications to establish safety protocols and ensuring the well-being of older individuals.Furthermore, broadening the pool of participants to include a more diverse and representative sample of older adults will enhance the generalizability of findings to a broader population.In essence, these additional studies on glutamine tolerability in older adults are indispensable for addressing existing knowledge gaps, refining recommendations, and guaranteeing the safety and efficacy of glutamine supplementation.
A noteworthy limitation of this study was the small number of volunteers who met the inclusion criteria for participation in this clinical investigation.Despite this limitation, the findings revealed that oral supplementation with EGln was safe for older adults.

Table 1 .
Plasma levels of amino acids in older adults before (day 0) and after (day 60) treatment with the effervescent glutamine formulation (EGln) or placebo (PL).Data are reported as means±SD of three analyses (n=12-13/group).*Po0.05,unpaired Student's t-test was used to compare the results between the EGln and placebo groups on days 0 and 60.

Table 2 .
Hematological parameters in older adults before (day 0) and after 30 (day 30) and 60 days (day 60) of daily supplementation with 12.4 g of effervescent glutamine (EGln group) or 12.4 g of maltodextrin (PL group).

Table 3 .
Continued.Data are reported as means±SD (n=15/group).*Po0.05 between day 60 and day 0, ANOVA with the Tukey test for multiple comparisons was used to evaluate variations in results within each group across days 0, 30, and 60.
Data are reported as means±SD (n=15/group).HbA1c: glycated hemoglobin A1c; AST: aspartate aminotransferase; ALT: alanine aminotransferase; CRP: C reactive protein.P40.05, ANOVA with the Tukey test for multiple comparisons was used to evaluate variations in results within each group across days 0, 30, and 60.