Efficacy of leucine-rich high protein supplementation on body composition and muscle function among older adults with sarcopenia: a randomized controlled trial

Abstract

Purpose

To evaluate the effect of leucine-rich high protein supplementation on body composition, muscle function and gene expression among older adults with or at risk of sarcopenia residing in Klang Valley, Malaysia.

Methods

A total of 47 participants were recruited into this 12-week double-blinded, randomized controlled trial. Intervention group consumed 2 sachets of high protein supplement daily contributing to 50.6 g of protein/day and 6 g of leucine/day. Control group received placebo supplement. Body composition was assessed via anthropometry measurements, bioelectrical impedance analysis (BIA), and magnetic resonance imaging (MRI) of left mid-thigh. Meanwhile, muscle function was assessed using short physical performance battery (SPPB). A total of 16 ml of full blood was collected pre- and post- study to assess participants’ health profiling and changes in gene expression as determined by mitochondrial RNA activity derived from the peripheral blood mononuclear cells. The measurements were performed at 0, 6, and 12 weeks.

Results

Participants were mostly women (89.4%) with a mean age of 69.3 ± 7.1 years. Repeated measures ANOVA showed no significant intervention effect in body composition (anthropometry measurements and BIA) as well as muscle function (SPPB score). However, genes responsible for adenosine triphosphate (ATP) production (GBA, MLYCD), cell proliferation (STAT5A) and DNA repair (BRCC3) were significantly up-regulated in intervention group (p < 0.05).

Conclusion

Leucine-rich high protein supplementation did not produce significant changes in body composition or muscle function in older adults with sarcopenia. However, it showed potential in improving gene expression. Further studies with a longer supplementation period and a larger sample size might be needed for noticeable changes, particularly for body composition.

Introduction

Sarcopenia is a word that derives from Greek, where sarx is flesh and penia means loss. European Working Group on Sarcopenia in Older People defines sarcopenia as a syndrome characterized by progressive and generalized loss of skeletal muscle mass and strength [1]. In the year 2014, the first-ever Asian Working Group for Sarcopenia (AWGS) defined sarcopenia as age-related loss of muscle mass, plus low muscle strength and or low physical performance which has been widely adopted [2].

A meta-analysis recently revealed that the global prevalence of sarcopenia by the definition of AWGS 2019 was 14% among individuals aged ≥ 18 years [3]. However, a few local studies [4–7] had shown that when different clinical definitions were applied, around 13–59% of Malaysian older adults aged 50 years and above suffered from sarcopenia, which were much higher than the aforementioned global prevalence. This can be attributed to several interrelated factors, including lower physical activity levels and inadequate protein intake [4]. If left untreated, sarcopenia can have detrimental consequences including functional decline [8], poor physical performance, and physical disability [9]. Longitudinal epidemiological studies demonstrated that there is an increased risk for disability as determined by instrumental activities of daily living (IADL) [10, 11] and falls [12, 13] among older adults with sarcopenia, which in turn impairs one’s quality of life [14].

A recent study found that branched-chain amino acids (BCAAs) was strongly associated with the muscle mass or strength in individuals aged ≥ 60 years [15]. Therefore, protein consumption has garnered a lot of attention in combating sarcopenia due to its ability to stimulate an increase in muscle protein synthesis [16] as well as its anti-inflammatory profile [17]. A higher protein intake from 1.0 to 1.5 g/kg/day with addition of leucine-enriched protein supplementation is recommended to enhance muscle strength [18]. A previous clinical trial demonstrated that older adults with sarcopenia can absorb orally administered leucine, leading to improvements in certain functional parameters such as walking time and respiratory muscle strength, indicating effective absorption and utilization [19]. Leucine (1.2–6 g/day) primarily enhances muscle function directly by stimulating muscle protein synthesis via the mTOR signaling pathway [20]. It also inhibits proteolysis, contributing to muscle mass maintenance [20]. Additionally, leucine may have systemic effects, such as stimulating insulin release, which can further support muscle anabolism [21]. A systematic review concluded that protein supplement, whether used alone or in conjunction with exercise, exhibited positive effects on muscle mass, muscle strength, and physical function in older adults with sarcopenia [22, 23].

Although studies related to protein supplementation on sarcopenia had been carried out for the past decade, most of them investigated the combined effect of protein and resistance exercise [7, 24–32] or even electrical muscle stimulation [33], rather than using protein supplement as a monotherapy. Some of the studies measured the skeletal muscle mass of the participants only by using BIA [34, 35]. However, BIA had been reported to show inaccuracies in assessing skeletal muscle mass in older adults [36]. Other objective measures such as magnetic resonance imaging biomarkers had been reported to be more accurate [37, 38]. One study only considered the physical and functional performance of participants without looking into changes in body composition [39], whilst the rest of the studies [40–42] did not delve into the interaction between protein supplementation and gene expression. Measuring gene expression offers deeper insights on the genetic factors involved in the development or progression of sarcopenia [43, 44]. Studies which did examine gene expression used T cells or muscle RNA expression rather than mitochondrial RNA (mRNA) activity [45–47], despite the fact that mitochondrial dysfunction has long been associated with sarcopenia of aging [48, 49].

While muscle biopsies remain the gold standard for assessing muscle-specific molecular changes, emerging evidence indicates that gene expression patterns in peripheral blood mononuclear cells (PBMCs) can reflect muscle function [50]. This supports the use of PBMCs as a surrogate model for skeletal muscle tissue in nutrigenomic studies [50]. In the present study, mRNA was extracted from PBMCs due to the minimally invasive nature of blood collection [51]. which is particularly important when working with older adults. Muscle biopsy procedures can be challenging in this population—especially among older female participants with low muscle mass—due to a higher risk of sampling failure [52]. Additionally, certain individuals may have contraindications to biopsy, such as a history of frequent falls or the use of anticoagulant medications [52].

Thus, the purpose of this study was to assess the efficacy of leucine-rich high protein supplementation on body composition via anthropometry measurements, bioimpedance analysis (BIA) and magnetic resonance imaging (MRI); muscle function via short physical performance battery (SPPB); and gene expression through mitochondrial RNA activity (Nanostring® gene expression analysis).

Methodology

Study design

A double-blinded randomized controlled trial (RCT) was adopted to investigate the efficacy of leucine-rich high protein supplementation, whereby participants in the intervention group were required to consume the supplements at 2 sachets a day for 12 weeks, whilst the control group took placebo supplements, also 2 sachets a day for the same duration of time. Outcomes were assessed at baseline, mid-point (week-6) and at the end of the trial (week-12). Participants of the study were selected using either convenience or purposive sampling, and were assigned to different groups via block randomization.

Sample size

With reference to Ten-Haaf et al. [53], the mean change in lean body mass from baseline to the end of the study differed by 0.11 ± 0.17 kg between the treatment and placebo groups. By inserting this mean difference into the RCT formula [54] taking into account 80% power and 95% confidence interval, a total of 42 participants was found to be sufficient. However, a high dropout rate of 50% was assumed due to possible gastrointestinal symptoms as well as taste issues. As a result, 32 participants were included in each of the intervention and placebo arms, bringing the final sample size to a grand total of 64 participants. On the other hand, the sub-sample for MRI subjects was determined based on a meta-analysis [55], where the changes in muscle fibre cross-sectional area (CSA) between the control and placebo group was 310 ± 260 µm². With the same RCT formula, it was found that 8 participants were needed in each arm to undergo thigh MRI. Again, by assuming a 50% of dropout rate, there would be 12 subjects in each arm, which brings the sample size to 24 participants. Nanostring® gene expression analysis was also performed on this subgroup of participants.

Study population

This RCT targeted senior community dwellers residing in the Klang Valley region within the state of Selangor, located in the central region of Malaysia. Participants were recruited from several Senior Citizen Centres (PAWE), the Outpatient Geriatric Clinic of Hospital Canselor Tunku Muhriz UKM (HCTM), non-government organizations like the Young Men's Christian Association (YMCA) of Kuala Lumpur, and a few of Malaysian worship places. In order to be eligible for this study, participants had to be Malaysians aged 60 years and above, with skeletal muscle index (SMI) of < 7.0 kg/m² for male and < 5.7 kg/m² for female, had a Mini Mental State Examination (MMSE) score more than 17, had no food security issue, able to eat and drink safely without modifying food texture, and were not actively participating in other interventional studies. Participants must not have poorly controlled diabetes, chronic kidney disease (Ckd) stage 3a and above, liver failure, malignancy, gastrointestinal diseases, thyroid diseases, severe malnutrition, or experience milk and/or soy intolerance or allergy.

Intervention

The experimental group received Resurge Gold 55 +, whilst the control group was given Resurge Placebo, of which both were provided by Uno Nutrition Sdn Bhd and Quantum Upstream Sdn Bhd. Participants of both groups started consuming the supplements after their baseline assessment for a total period of 12 weeks. The supplement, whether intervention or placebo, was taken twice daily after meals, either after breakfast, lunch, or dinner according to their preference. It was packed in sachet form to allow easier administration. Each sachet of the intervention supplement provides 25.3 g of protein as indicated in the nutritional facts displayed in the Table 1 (information provided by the product company). Besides, each sachet of the intervention supplement also provides a total of 3.00 g of total leucine of which approximately 2.56 g is naturally present in the whey protein and 0.44 g is added as free leucine during product reformulation (information provided by the product company). The additional 0.44 g of leucine was blended with other ingredients during the formulation stage, following standard industrial fortification practices. It is not feasible to open sachets and add leucine individually in any bulk production setting. The product tested in this study was provided to participants in sealed, ready-to-use sachets identical to those commercially available, and the authors did not manually add leucine at any point (information confirmed with the product company). Table 2 displays the amino acid profile of the supplement. In contrast, one sachet of placebo supplement contains 3.3 g of protein (leucine 0 g).

Table 1.

Nutrition content of resurge gold 55 + and resurge placebo

Per serving (serving size: 48 g)	Resurge gold 55 +	Resurge placebo
Energy (kcal)	176	166
Fat (g)	3.4	3.9
Saturated fatty acid (g)	1.3	0.5
Carbohydrate (g)	9.8	26.5
Fibre (g)	2.4	5.9
Protein (g)	25.3	3.3
Leucine (g)	3.0	0.0
Vitamin D3 (IU)	796.4	3.2
Vitamin K (µg)	12.0	0.0
Choline (mg)	56.0	0.0

Table 2.

Amino acid profile of the protein supplement received by the experimental group

Amino acid profile	Per 100 g (g)
Aspartic acid	5.8
Threonine	3.3
Serine	2.2
Glutamic acid	8.9
Glycine	1.0
Alanine	2.5
Valine	3.3
Isoleucine	3.5
Leucine	6.3
Tyrosine	1.4
Phenylalanine	1.7
Histidine	0.9
Lysine	4.4
Arginine	1.2
Proline	3.0
Cystine	1.1
Methionine	1.1
Tryptophan	0.9
BCAA	13.2
EAA	27.5
Non-EAA	24.9

The detailed nutritional content of both the intervention supplement and the placebo supplement is presented in Table 1. One sachet of intervention supplement provided 176 kcal while the placebo supplement provided 166 kcal. The caloric content of both supplements was derived from the carbohydrate, fibre, protein and fat as shown in Table 1. In addition to macronutrients, the intervention supplement also contained other components such as vitamin D3 and choline, which were either present in negligible amounts or entirely absent in the placebo supplement. (Table 1). By taking 2 sachets daily, participants from intervention group would each receive a total of 50.2 g protein, which meets at least 87% of Malaysian Recommended Nutrient Intake (RNI), and 20 µg of vitamin D3, which meets 100% of Malaysian RNI [56]. There is no established Malaysian RNI nor international consensus on optimal leucine intake in older adults, but PROT-AGE recommended an intake of 2.5–2.8 g of leucine per meal [57]. However, it is important to note that these RNI and recommendation targeted healthy older adult population, which infers a higher need among older adults with or at risk of sarcopenia. Indeed, previous randomized controlled trial have shown that supplementation with 6 g/day of leucine can have beneficial effects on muscle health and may be considered for the treatment of sarcopenia in older individuals [19].

Each sachet of the supplement [48 g] needed to be mixed well with 160 ml of lukewarm or cold water. Participants were reminded not to treat the supplement as meal replacement. A written guideline on preparing the supplement drink was provided to them. Regular WhatsApp texts were sent to all participants to remind them to complete their prescribed dosage and inform about their upcoming mid- (week-6) and endpoint- (week-12) assessment dates. Apart from that, the participants were also asked for feedback on their consumption progress including any possible side effects they may experience, as well as their liking towards the supplement. During their visit for midpoint assessment, investigator would confirm with participants about the number of supplements left before handing out the second half of the prescribed dosage. At endpoint assessment, participants were asked to bring along their remaining supplements for compliance evaluation. Participants’ compliance rate was calculated based on the number of packets they took, with a higher percentage indicating better compliance.

Trial registration number

This trial has been registered retrospectively under Australian New Zealand Clinical Trials Registry (ACTRN: ACTRN12624000277549).

Data collection

Data collection was carried out between 15th December 2021 and 19th February 2024 by a group of trained fieldworkers. Upon screening for eligibility, participants with SMI below normal range went through blood test, where a total of 8 ml of fasting venous blood was collected for health screening purpose. Participants who fit both criteria (SMI and MMSE) were invited to take part in the study. They gave informed consent before being officially recruited. Assessments of participants were conducted at baseline, week-6 and week-12. At baseline, participants were interviewed for their sociodemographic data and medical history. Their sarcopenia status was determined by looking at SMI, handgrip strength of dominant hand and 6-m gait speed test. Participants were measured for primary outcomes namely body composition via BIA, anthropometry measurements and MRI, and muscle function via SPPB. Other secondary measurement was mRNA gene expression, which was also assessed at baseline. A subsample of each arm was further selected randomly using a computer-generated randomisation sequence for MRI of left mid-thigh. The subgroups were matched for their gender, age and sarcopenia status to reduce potential confounding and ensure group comparability. The same subgroup would also have an additional 8 ml of venous blood drawn for gene expression analysis. All measurements were repeated at week-6 and week-12 except for MRI and gene expression analysis, which were only conducted at week-12.

Measurements and outcomes

Demographics data

Socio-demographic questionnaire was used to obtain participants’ information including basic demographics, health behavior, oral nutrition supplement consumption, concurrent participation in other interventional studies, implant status, and claustrophobia status. A separate medical history form was administered to gather information regarding participants’ health condition.

Sarcopenia status

Sarcopenia status of participants was assessed based on the definition of AWGS 2019 [58]. A sarcopenia determinant form which consists of three domains, namely SMI, handgrip strength and 6-m gait speed test was used. Participants’ SMI value was obtained via InBody 270 body composition analyzer. The skeletal muscle mass cut-off points (skeletal mass index, SMI) are < 7.0 kg/m² for men and < 5.7 kg/m² for women. Handgrip strength for dominant hand was determined by using Jamar Plus digital hand dynamometer FAB12-0604. Poor handgrip strength is defined as < 26 kg for males and < 18 kg for females. In 6-m gait speed test, participants were instructed to walk at a normal speed, and the best time was recorded. Poor gait speed is defined as ≤ 0.8 m/s for both men and women. By combining the results of the three domains, participants were then classified into pre-sarcopenia, sarcopenia or severe sarcopenia stage.

Body composition

Body composition with emphasis on SMI, body weight, body mass index (BMI), body fat percentage and skeletal muscle mass, was assessed using InBody 270 body composition analyzer from South Korea.

Anthropometry measurements

Anthropometry measurements taken were mid-upper arm circumference (MUAC), mid-thigh circumference and calf circumference. MUAC was determined by measuring participants’ left arm, at the mid-point between their olecranon process and acromion. A reading less than 28.6 cm in men and less than 27.5 cm in women indicate a state of sarcopenia [59]. Mid-thigh circumference was assessed by measuring the subject’s left thigh at the midpoint between the inguinal crease and the proximal border of the patella. As for calf circumference, the reading was obtained by measuring the widest part of participants’ left calf. 34 cm and 33 cm are the respective cut-off point of calf circumference for men and women [58, 60]. A Lufkin tape was used to take all the said measurements. Every measurement was repeated twice and the average reading was calculated.

Skeletal muscle mass

Thigh imaging was used as a proxy measure for participants’ whole-body skeletal muscle mass, as there was a strong correlation between thigh skeletal muscle and whole-body skeletal muscle measurements [61]. Imaging was conducted at the mid-level of left thigh for 22 sub-samples of participants via Siemens Magnetom Skyra 3 T MRI machine. The data acquisition parameters applied in this study adhere to the protocol outlined in a previous study [62]. Changes in muscle cross-sectional area, total cross-sectional area and proton density fat fraction (PDFF) were among the parameters monitored in this trial.

Physical performance (muscle function)

Short Physical Performance Battery, SPPB [63] was used to assess participants’ physical fitness level. This series of timed tests comprise of standing balance, walking speed and chair stand tests [64]. All 3 scores were added up and a score that is 9 or below indicates low physical performance.

mRNA gene expression

Peripheral blood mononuclear cells (PBMCs) were isolated from participants’ whole blood via density gradient centrifugation. RNAs were then extracted from the PBMCs samples using Geneaid Total RNA Mini Kit. Samples which did not meet the requirement would be cleaned up using Geneaid RNA cleanup kit. Once the RNA samples were ready, they would undergo a 16-h hybridization process, followed by a 6-h analysis using nCounter SPRINT Profiler. The final results were interpreted by nSolver™ Analysis Software to look at the transcriptomic pathway in relation to sarcopenia.

Statistical analyses

Data were analyzed with SPSS statistical software, version 26.0 (IBM) by adhering to intention-to-treat principle. A confidence interval of 95% was applied, and significant difference was defined by p < 0.05, with a power of 0.80. Missing data were addressed using mean/mode imputation. Descriptive analysis was performed. The normality of data was determined by kurtosis test. Repeated measures ANOVA was conducted to compare within-subject changes, between-group changes as well as intervention effect in terms of body composition, muscle function, and mitochondrial RNA activity. Covariates including age, gender, education, and physical activity levels were taken into consideration in the analyses.

Post-trial care

Participants would not have access to the trial supplements after the end of study, but they would be informed about the availability of similar products in the market. Apart from that, participants would also receive a brief counseling on how to improve their muscle mass and function through daily dietary and exercise recommendations.

Results

Initially, a total of 69 participants were recruited and randomized into intervention and placebo groups. However, 22 of them dropped out before week-6 assessment and another 4 before week-12 assessment, leaving only 43 participants who completed the entire trial. Given the small sample size, intention-to-treat analysis was applied to minimize possible bias. For mid-thigh MRI, 12 participants were randomly selected from experimental and placebo groups respectively. Six participants from each of these MRI subgroups were further selected to undergo mRNA gene expression. A CONSORT flow chart is shown in Fig. 1.

Fig. 1.

CONSORT study flow chart

Baseline characteristics

As depicted in Table 3, the mean age of participants was 69.3 ± 7.1 years. Majority of the participants were females (89.4%), Chinese (68.1%) and married (55.3%). Most of them received tertiary education (51.1%). Majority of the participants had a household income of less than RM5000/month (83.0%). Most of them were diagnosed with hyperlipidemia (63.8%). Out of 47 participants, 57.4% had sarcopenia while 21.3% had severe sarcopenia and 21.3% had pre-sarcopenia. The baseline characteristics of participants from both groups did not differ after randomization.

Table 3.

Baseline characteristics between intervention and placebo group participants [presented as mean ± standard deviation or n (%)]

Parameter	Total (n = 47)	Intervention (n = 25)	Placebo (n = 22)	p-valuea
Sociodemographic
Age (60–90)	69.3 ± 7.1	68.8 ± 6.5	69.7 ± 7.9	0.674
Gender				0.753
Men	5 (10.6)	3 (12.0)	2 (9.1)
Women	42 (89.4)	22 (88.0)	20 (90.9)
Ethnicity				0.336
Malay	12 (25.5)	6 (24.0)	6 (27.3)
Chinese	32 (68.1)	16 (64.0)	16 (72.7)
Indian	3 (6.4)	3 (12.0)	0 (0)
Marital status				0.333
Single	8 (17.0)	6 (24.0)	2 (9.1)
Married	26 (55.3)	14 (56.0)	12 (54.5)
Divorced	2 (4.3)	0 (0)	2 (9.1)
Widowed	11 (23.4)	5 (20.0)	6 (27.3)
Educational level				0.326
No formal education	1 (2.1)	1 (4.0)	0 (0)
Primary	6 (12.8)	2 (8.0)	4 (18.2)
Secondary	16 (34.0)	7 (28.0)	9 (40.9)
Tertiary	24 (51.1)	15 (60.0)	9 (40.9)
Monthly household income				0.315
< USD 1,06	39 (83.0)	19 (76.0)	20 (90.9)
≥ USD 1,060	8 (17.0)	6 (24.0)	2 (9.1)
Medical history
Hypertension				0.769
Yes	16 (34.0)	9 (36.0)	7 (31.8)
No	31 (66.0)	16 (64.0)	15 (68.2)
Hyperlipidemia				0.570
Yes	30 (63.8)	15 (60.0)	15 (68.2)
No	17 (36.2)	10 (40.0)	7 (31.8)
Diabetes				0.563
Yes	7 (14.9)	3 (12.0)	4 (18.2)
No	40 (85.1)	22 (88.0)	18 (81.8)
Health profile
Fasting blood sugar (mmol/L)	5.5 ± 1.2	5.3 ± 1.0	5.7 ± 1.4	0.319
Estimated glomerular filtration rate (eGFR) (> 90 mL/min/1.73 m2)				0.887
Stage 1	20 (42.6)	9 (36.0)	11 (50.0)
Stage 2	27 (57.4)	16 (64.0)	11 (50.0)
Sarcopenia status				0.662
Pre-sarcopenia	10 (21.3)	4 (16.0)	6 (27.3)
Sarcopenia	27 (57.4)	16 (64.0)	11 (50.0)
Severe sarcopenia	10 (21.3)	5 (20.0)	5 (22.7)
Weight	50.4 ± 7.2	50.9 ± 7.8	49.8 ± 6.0	0.787

^aIndependent t-test, not significant at p > 0.05

Efficacy of leucine-rich high protein supplementation

Body composition

In terms of body weight, both groups demonstrated a statistically significant increase after 12 weeks. The intervention group increased from 50.9 ± 7.7 kg at baseline to 51.7 ± 7.7 kg post-intervention (p = 0.022), while the placebo group increased from 49.8 ± 6.0 kg to 50.8 ± 6.1 kg (p = 0.006). However, the between-group difference was not statistically significant (p = 0.667) (results not tabulated). As presented in Table 4, there was no significant effects in terms of MUAC, calf circumference and mid-thigh circumference measurements between intervention and placebo groups after taking the supplements. Both groups showed an increase in terms of BMI after 12 weeks (p < 0.001) but the difference between groups was not statistically significant. Similar pattern was observed in the total cross-sectional area of left mid-thigh through MRI, where although both groups showed increment after 12 weeks of supplementation (p = 0.022), there was no statistical difference between them. Figure 2 illustrates the percentage mean change in muscle cross-sectional area and PDFF in the intervention and placebo groups before and after the trial.

Table 4.

Effectiveness of leucine-rich high protein supplementation on body composition as assessed using BIA, anthropometry measuremets and MRI [presented as mean ± standard deviation]

Parameter	Intervention (n = 25)	Placebo (n = 22)	Group effect			Time effect			Group × Time effect
			P	Partial-Eta squared	Power	P	Partial-Eta squared	Power	P	Partial-Eta squared	Power
BIA
SMI (kg/m2)
Baseline	5.2 ± 0.6	5.3 ± 0.5	0.598	0.006	0.081	0.055	0.065	0.551	0.633	0.009	0.112
6th week	5.3 ± 0.6	5.3 ± 0.6
12th week	5.2 ± 0.6	5.3 ± 0.5
Anthropometry measurements
BMI (kg/m2)
Baseline	21.6 ± 3.0	21.8 ± 3.1	0.781	0.002	0.059	< 0.001	0.187	0.985	0.808	0.005	0.083
6th week	21.9 ± 3.1	22.2 ± 3.1
12th week	21.9 ± 3.0	22.2 ± 3.1
MUAC (cm)
Baseline	24.1 ± 2.4	24.4 ± 2.5	0.591	0.006	0.083	0.321	0.025	0.247	0.973	0.001	0.054
6th week	24.1 ± 2.4	24.6 ± 2.5
12th week	24.4 ± 2.2	24.7 ± 2.3
Calf circumference (cm)
Baseline	31.9 ± 1.7	32.4 ± 2.8	0.574	0.007	0.086	0.503	0.013	0.125	0.394	0.019	0.164
6th week	31.9 ± 1.9	31.9 ± 3.1
12th week	31.8 ± 1.5	32.3 ± 2.5
Mid-thigh circumference (cm)
Baseline	40.5 ± 3.4	41.3 ± 4.6	0.447	0.013	0.117	0.598	0.011	0.127	0.787	0.005	0.083
6th week	39.8 ± 4.4	41.0 ± 4.3
12th week	40.5 ± 3.9	41.0 ± 4.9
MRI
Muscle cross sectional area (cm2)
Baseline	74.8 ± 11.9	77.5 ± 16.2	0.734	0.006	0.062	0.159	0.092	0.287	0.787	0.004	0.058
12th week	78.3 ± 16.5	79.9 ± 17.8
Total cross sectional area (cm2)
Baseline	130.7 ± 23.1	136.2 ± 30.2	0.532	0.019	0.093	0.022	0.224	0.652	0.692	0.008	0.067
12th week	139.3 ± 20.7	148.2 ± 38.8
Percentage of total cross sectional area (%)
Baseline	58.2 ± 11.1	58.8 ± 14.1	0.999	0.000	0.050	0.041	0.184	0.548	0.515	0.020	0.097
12th week	56.8 ± 12.2	56.2 ± 13.2
Average PDFF
Baseline	12.6 ± 2.9	12.9 ± 3.1	0.593	0.014	0.081	0.668	0.009	0.070	0.362	0.040	0.144
12th week	12.1 ± 2.5	13.1 ± 3.7

BMI, body mass index; MUAC, mid-upper arm circumference; MRI, magnetic resonance imaging; PDFF, proton density fat fraction

^aIndependent t-test, not significant at p > 0.05

Fig. 2.

Percentage mean change of muscle cross-sectional area a and proton density fat fraction (PDFF) b between intervention and placebo groups

Muscle function

The results of SPPB are shown in Table 5. Even though both groups showed improvement in 4-m gait speed (p < 0.001) and repeated chair stand (p = 0.002 and p = 0.012 for time and total score respectively) tests after 12 weeks, no significant difference was found between groups.

Table 5.

Effectiveness of leucine-rich high protein supplementation on muscle function as assessed using SPPB [presented as mean ± standard deviation]

Parameter	Intervention (n = 25)	Placebo (n = 22)	Group effect			Time effect			Group × Time effect
			P	Partial-Eta squared	Power	P	Partial-Eta squared	Power	P	Partial-Eta squared	Power
Total balance score
Baseline	3.4 ± 0.9	3.7 ± 0.8	0.943	0.000	0.051	0.980	0.000	0.052	0.099	0.051	0.457
6th week	3.6 ± 1.0	3.5 ± 1.1
12th week	3.6 ± 0.9	3.5 ± 0.8
4-m gait speed test (m/s)
Baseline	1.0 ± 0.2	1.0 ± 0.3	0.683	0.004	0.069	0.001	0.143	0.936	0.953	0.001	0.057
6th week	1.1 ± 0.3	1.1 ± 0.3
12th week	1.1 ± 0.3	1.1 ± 0.3
Total gait speed score
Baseline	3.7 ± 0.5	3.6 ± 1.0	0.561	0.008	0.089	0.408	0.020	0.202	0.779	0.006	0.088
6th week	3.8 ± 0.7	3.7 ± 0.9
12th week	3.7 ± 0.7	3.6 ± 1.0
Repeated chair stand test (s)
Baseline	14.0 ± 9.9	11.2 ± 5.3	0.595	0.006	0.082	0.002	0.162	0.895	0.157	0.043	0.328
6th week	9.5 ± 3.9	10.1 ± 4.3
12th week	8.5 ± 3.7	8.9 ± 2.6
Total RCST score
Baseline	3.0 ± 1.3	3.4 ± 0.9	0.306	0.023	0.173	0.012	0.094	0.773	0.617	0.011	0.127
6th week	3.3 ± 1.0	3.5 ± 1.0
12th week	3.4 ± 1.2	3.7 ± 0.6
Overall SPPB score
Baseline	10.2 ± 2.1	10.6 ± 2.2	0.845	0.001	0.054	0.157	0.040	0.383	0.310	0.026	0.254
6th week	10.7 ± 2.2	10.6 ± 2.3
12th week	10.8 ± 2.5	10.8 ± 1.8

RCST, repeated chair stand test; SPPB, short physical performance battery

^aIndependent t-test, not significant at p > 0.05

mRNA genetic expression

Figure 3 shows the differential expression of mRNA genes in intervention group versus placebo group after 12 weeks of supplementation. Genes including Glucocerebrosidase (GBA), Malonyl-CoA Decarboxylase (MLYCD), Signal Transducer and Activator of Transcription 5A (STAT5A) and BRCC3 were found to be significantly up-regulated in intervention group compared to placebo group (p < 0.001).

Fig. 3.

Differential expression in intervention group versus placebo group (*The top 20 genes included GBA-mRNA, STAT5A-mRNA, BRCC3-mRNA. MLYCD-mRNA, NADK2-mRNA, SREBF1-mRNA, PIK3R1-mRNA, HLA-DRB1-mRNA, ITK-mRNA, LDHB-mRNA, TLR2-mRNA, CD68-mRNA, NADK-mRNA, HK1-mRNA, IMPDH1-mRNA, CD3D-mRNA, HEXA-mRNA, BCL2A1-mRNA, CD3E-mRNA and KYNU-mRNA)

Adverse events and compliance rate

During midpoint follow-up (week 6), 68.2% (n = 15) of the participants from experimental group reported feeling normal after high protein supplementation, whilst 18.2% (n = 4) of them complained of early satiety which hindered them from taking their main meals normally. Another three participants complained about gastrointestinal related issues, namely constipation (4.5%), bloating (4.5%) and flatulence (4.5%). At the same time, nearly half (42.9%) of the participants from placebo group experienced discomfort after taking the placebo supplements, including early satiety (14.3%), soft stool (14.3%), diarrhea (14.3%), bloating (9.5%) and flatulence (4.8%). Nevertheless, participants’ condition from both groups improved during endpoint follow-up (week 12), where only 1 participant complained of constipation (4.5%) and another of early satiety (4.5%) respectively in experimental group, while in placebo group, only 2 participants complained of either having soft stool (4.8%) or bloating issue (4.8%). Apart from that, out of 43 participants who completed the trial, 65.1% (n = 28) had an 100% compliance rate, 9.3% (n = 4) had at least a 90% compliance rate, while 25.6% (n = 11) had a compliance rate between 50 and 83% throughout the trial.

Discussion

This study aimed to assess the efficacy of leucine-rich high protein supplementation on body composition, muscle function and gene expression. Mid-thigh MRI findings revealed that there was a time effect in terms of total CSA in both groups. Nonetheless, total CSA might not be a good indicator of muscle mass gain, as it is affected by the presence of other tissues such as fat and fibrosis [42]. Otherwise, no significant difference between groups was observed in muscle CSA, total CSA as well as average PDFF. This aligns with the findings of a previous study, where there were no between-group differences in changes in quadriceps CSA among those who consumed supplements regardless of their protein types [65]. In contrast to previous findings [42], the present study did not observe a significant improvement on muscle function as assessed using SPPB score following high protein supplementation, possibly due to the absence of a structured exercise prescription. Indeed, a meta-analysis demonstrated that nutritional supplementation did not help to improve SPPB score unless it was coupled with a physical exercise program [66]. Nevertheless, when looking at the SPPB domains separately, it was noticed that 4-m gait speed improved significantly in both groups on week 6 of supplementation, but deteriorated on week 12. Studies on the effect of high protein supplementation on gait speed yielded mixed results in the past [17, 34, 67, 68]. An important consideration in the effectiveness of protein intake on muscle mass is not only total daily intake but also achieving a sufficient protein dose per meal to overcome anabolic resistance and maximize muscle protein synthesis. Older individuals typically require approximately 0.4 g/kg of high-quality protein per meal (or 25–30 g/meal) to optimally stimulate muscle protein synthesis [69]. Based on the participants’ mean body weight (50.4 kg) and the protein content per sachet (25.1 g), the mean relative protein intake was approximately 0.50 g/kg per meal. This exceeds the recommended threshold of 0.4 g/kg/meal for older adults, suggesting that the supplemented protein dose was sufficient to stimulate muscle protein synthesis. Further study should focus on the comprehensive dietary profile of older adults, including total protein intake, protein distribution across meals, and the quality and source of protein, to better understand their contributions to muscle health and the management of sarcopenia. This approach may help identify specific nutritional patterns that are most effective in overcoming anabolic resistance and preserving muscle mass in aging populations.

Apart from that, both groups also showed an improvement in terms of repeated chair stand test over time, but there was no between-group difference. The findings were consistent with the PROVIDE study in the first part, but contrasted with it in the second part [42]. This again could be due to the threshold of compliance rate, of which our study accepted a compliance rate of as low as 50%, while the said study only considered participants with a minimum 71% compliance rate. A lower compliance rate translates into lower nutrition uptake from the supplements, hence resulting in a lower stimulation of acute muscle protein synthesis [42]. Although direct evidence linking acute stimulation of muscle protein synthesis to chronic changes in total muscle mass is not presented in the current study, the observed improvement in the repeated chair stand test following protein supplementation presumed to reflect acute anabolic responses may suggest functional outcomes associated with long-term muscle adaptation. This assumption is supported by previous research, such as Bauer et al. [42]. However, the limitations of extrapolating acute responses to long-term outcomes, particularly in sarcopenic populations should be acknowledged. Longitudinal studies would be more appropriate to confirm this relationship.

On the other hand, both experimental and control groups exhibited an increase in BMI over time. This contradicted with a previous local study which showed a reduction in participants receiving protein supplementation [7]. The reason could be that the supplements used in our study have a much higher energy content (332 kcal/day for control; 348 kcal/day for experimental) compared to the aforementioned study (87 kcal/day for male participants; 174 kcal/day for female participants), resulting in an overall higher daily energy intake. Nonetheless, there was no significant difference in BMI between experimental and control groups. This again contradicted with a systematic review, which found that leucine supplementation significantly increased gain in body weight and BMI [64]. However, it is noteworthy that the said review included studies which recruited healthy older adults without sarcopenia, thus the findings might not be comparable. In fact, a more recent systematic review concluded that whey protein supplementation did not have a significant effect on BMI and body weight in patients with sarcopenia [17]. Next, current findings align with a previous systematic review which concluded that vitamin D plus protein supplementation had no effect on muscle mass of patients with sarcopenia [65]. This is most probably due to the absence of exercise prescription, as studies actually demonstrated the beneficial effect of exercise, either combined with protein supplements or as a monotherapy, in helping to increase muscle mass among older adults with sarcopenia [7, 27, 32].

Aging is known to be associated with impaired mitochondrial function [49]. Our study is the first of its kind in the region to look into the effect of high protein supplementation on sarcopenia in terms of mitochondrial RNA activity. Emerging evidence suggests a correlation between gene expression patterns in PBMCs and those in skeletal muscle. This supports the use of PBMCs as a surrogate model for skeletal muscle tissue in nutrigenomic studies [50]. Genes which were found to be up-regulated in the experimental group following protein supplementation include GBA, MLYCD, STAT5A, and BRCC3. Both GBA and MLYCD are genes responsible for ATP production, where GBA involves in lipid (glucocerebroside) breakdown while MLYCD provides instructions for making Malonyl-CoA decarboxylase, an enzyme that involves in fatty acid metabolism [70]. Higher ATP production is most likely fueled by fatty acid metabolism as demonstrated by the up-regulation of the said genes [71]. This was demonstrated as the gene expression of STAT5A, which acts as a transcription factor at cell nucleus that plays a role in cell proliferation [72], was also up-regulated among participants following high protein supplementation. Thus, it can be concluded that GBA, MLYCD and STAT5A promote muscle anabolism by enhancing muscle protein synthesis, resulting in increased muscle mass, and thus a reduced risk of sarcopenia. Meanwhile, BRCC3 is a gene that involves in DNA repair [73]. It enables the repair mechanism needed for DNA mutation to occur at cellular level [74, 75]. The up-regulation of BRCC3 gene means that the repairing rate had also increased, thereby contributing to the reversibility of ageing muscle cells. As mentioned earlier, apart from its role in muscle anabolism, NADP + also involves in antioxidant defense as well as muscle regeneration. Along with STAT5A, which promotes cell proliferation, these 3 genes help reduce muscle breakdown, minimizing muscle catabolism, thereby preserving muscle mass and lowering the risk of sarcopenia. The findings of mRNA gene expression align with the MRI finding, where the participants receiving leucine-rich high protein supplementation demonstrated a higher percentage mean change in terms of their thigh muscle cross-sectional area [42]. Figure 4 illustrates the relationship between the up-regulation of the aforementioned genes and sarcopenia.

Fig. 4.

Up-regulation of genes and their relation to sarcopenia

Apart from that, the levels of additional substances such as vitamin D, vitamin K, and choline in the intervention supplement are considerably lower than the doses typically examined in supplementation studies targeting sarcopenia. For example, vitamin D supplementation at higher doses (10,000 IU/day) has shown only minimal effects on sarcopenia-related parameters [76], while intervention studies on vitamin K have not yet confirmed a causal relationship with improvements in muscle or bone metabolism [77]. Similarly, low choline intake has been associated with diminished strength and lean mass gains in older adults [78]. Taken together, the amounts of these substances in the intervention supplement are insufficient to produce measurable effects on body composition or muscle function in older adults with sarcopenia. According to the study findings on renal function tests, only serum urea level showed a significantly greater positive percentage mean change in the experimental group receiving leucine-rich high protein supplementation compared to the control group. However, no significant changes were observed in other key markers of renal function such as serum creatinine or estimated glomerular filtration rate (eGFR) (results not shown). These results suggest that leucine-rich high protein supplementation does not substantially increase renal workload in older adults. Nonetheless, further studies with longer follow-up and comprehensive renal assessments are warranted to confirm its long-term renal safety.

There are several limitations in this study. Firstly, the use of convenience sampling has limited the study’s ability to extrapolate the findings to the broader older adults population in Malaysia. This limitation is exacerbated by the fact that participant selection was restricted to Klang Valley region. Secondly, the study did not exclude participants with pre-sarcopenia condition, therefore posted a risk of diluting the possible positive effects of the supplements as the changes might not be significant in them. Despite these weaknesses, the indisputable strength of our study lies in its examination of the effect of supplementation alone on sarcopenia through both MRI and mRNA analyses.

Conclusion

Leucine-rich high protein supplementation did not produce significant changes in body composition or muscle function in older adults with sarcopenia. However, it has the potential to up-regulate mRNA activity. A supplementation period longer than 12 weeks may be required for significant improvements in body composition and muscle function. Nonetheless, the findings of this study provide a novel insight prompting future investigation into the precise pathways through which protein supplementation is involved in mRNA regulation.

Funding

Open access funding provided by The Ministry of Higher Education Malaysia and Universiti Kebangsaan Malaysia. This study was funded by UNO Nutrition Sdn. Bhd. and Quantum Upstream Sdn. Bhd. (Grant no.: NN-2019-098).

Data availability

No additional unpublished data are available hitherto.

Declarations

Conflict of interest

The authors declare that they have no competing interests that are relevant to the content of this article. None of them was being employed by either UNO Nutrition Sdn. Bhd. Or Quantum Upstream Sdn. Bhd. at the time of study.

Ethical approval

This study received approval from the Universiti Kebangsaan Malaysia Research Ethics Committee (JEP-2019-208). All procedures performed in this study involving human participants were conformed to the Declaration of Helsinki.

Consent to participate

Written informed consent was obtained from all participants prior to their involvement in the study. Participation was solely on voluntary basis, and participants were explained about their right to opt out of the study at any time for any reason, without having to face any consequences or penalties. Likewise, the research team reseerved the right to withdraw any participants from the study if them deemed it to be in his or her best interest.