Effect of cysteine-rich whey protein (Immunocal®) supplementation in combination with resistance training on muscle strength and lean body mass in non-frail elderly subjects: A randomized, double-blind controlled study

Abstract

Objectives

The purpose of the present study was to examine the effect of a cysteine-rich whey protein (Immunocal®) supplementation in combination with resistance training on muscle strength and lean body mass (LBM) in elderly individuals. We hypothesized that the cysteine-rich whey protein (Immunocal®) group would experience a greater increase in muscle strength and lean body mass versus the control group (casein).

Design

Randomized double-blind controlled intervention study.

Setting

Institut de Recherches Cliniques de Montréal in Montreal, Canada.

Participants

Ninety-nine non-frail elderly subjects were recruited.

Intervention

Participants were randomly assigned into two groups. The experimental group received a cysteine-rich whey protein isolate (Immunocal®) (20 g/day) and the control group received casein (20 g/day) during a 135-day period. In addition, both groups performed the same resistance training program (3 times per week).

Measurements

Body composition (DXA) and muscle strength (leg press) were measured.

Results

Of the 99 recruited participants, 84 completed the 135-day study period. Of these, 67 subjects (33 in the casein group and 34 in the Immunocal® group) complied and used at least 80 % of the study product and completed at least 80 % of their training sessions. Results in this selected group show an increase in all three muscle strength variables (absolute, normalized by BW and by LBM) by 31.0 %, 30.9 % and 30.0 %, respectively in the casein group as well as 39.3 %, 39.9 % and 43.3 %, respectively in the Immunocal® group after the intervention (p < 0.05). The increases in muscle strength favored Immunocal® versus casein by approximately 10 % when expressed in kg per kg BW and in kg per kg LBM (p < 0.05). No significant changes were found between pre-and-post intervention in both groups for total LBM.

Conclusions

Our findings showed increases in muscle strength in both groups after resistance training, however, significant additional increases were observed in muscle strength with the addition of a cysteine-rich whey protein (Immunocal®) versus casein.

Introduction

Aging is typically associated with a decrease in skeletal muscle mass and muscle strength, which contributes decisively to disability in old age, an increase in the risk of falls and the loss of quality of life (1, 2). Muscle atrophy, weakness and functional impairment may already start in the fourth decade of life with a muscle strength loss of about 1 % per year, which then accelerates with each passing decade (3).

Resistance training can increase muscle mass and muscle strength in older adults even into the ninth decade of life (4). Moreover, an inadequate diet and especially a diet low in protein appear to be associated with a decrease in muscle mass and muscle strength (5, 6, 7). Several authors have even suggested that elderly individuals should consume more protein than the recommended daily allowance (RDA) of 0.8g kg^-1 d^-1 (8, 9, 10). It is now recommended to incorporate resistance training programs and an adequate protein intake for the maintenance of muscle mass and muscle strength in older individuals (11).

An increasing body of evidence has suggested that aging-related degenerative processes are mediated, at least in part, by oxidative stress (12, 13). Evidence from different fields collectively suggested that the age-related increase in oxidative stress may result from a progressive decrease in post-absorptive cysteine concentrations that is triggered by a vicious cycle involving decreasing glutathione concentrations as well as a progressive decrease in autophagic protein catabolism and amino acid homeostasis (14). Thus, targeting oxidative stress could be an important factor in preventing and/or treating age-related degenerative processes. In support of this concept, there is evidence to suggest that the production of oxidative stress could affect contractile function and muscle strength (15, 16).

Immunocal® is a whey protein supplement with a unique undenatured preparation that contains large quantities of cystine, a precursor of cysteine. Supplementation of the free amino acid L-cysteine alone does not appear to be effective in raising glutathione levels (17). However, since cystine is resistant to trypsin proteolysis, this allows it to freely circulate throughout the bloodstream to the target cell. Cystine is then readily reduced to two cysteine molecules which can serve as important precursors for de novo glutathione synthesis. Accordingly, previous studies have shown an increase in glutathione levels with Immunocal® supplementation (18, 19, 20). Furthermore, in a clinical trial, supplementation of a cysteine-rich undenatured whey protein was found to improve skeletal muscle function (18). That is, 3 months of Immunocal® supplementation significantly increased peak power and 30 sec work capacity in a whole leg isokinetic cycle test when compared to the control group (casein) in young healthy subjects (18). It should be noted that Immunocal® is included in the Physician’s Desk Reference (PDR) and the Compendium of Pharmaceuticals and Specialties (CPS).

In view of the fact that resistance training seems to be the best documented method for increasing muscle strength and/or muscle mass in humans, it is of special interest to determine whether cysteine-rich protein supplementation may cause a further improvement on top of the effects of resistance training in older adults. Therefore, the purpose of the present study was to examine the effect of a cysteine-rich whey protein (Immunocal®) supplementation in combination with resistance training on muscle strength and lean body mass (LBM) in elderly individuals. We hypothesized that the cysteine-rich protein (Immunocal®) group would experience a greater increase in muscle strength and lean body mass versus the control group (casein).

Methods

Subjects

This double-blind randomized controlled study recruited 99 sedentary non-frail elderly subjects aged between 65 and 88 years old, of which 76 were female and 23 male. Participants were randomly assigned into two groups. The experimental group received a cysteine-rich whey protein isolate (Immunocal®) and the control group received casein. In addition, all subjects performed the same resistance training program. Participants were recruited in the study using advertisements in local papers and Montreal communities. Subjects were included in the study if they met the following criteria: 1) aged 65 years or older, 2) estimated glomerular filtration rate above 45 ml/min, 3) a body mass index (BMI) between 18.5-29.9 kg/m², 4) sedentary (< 2 hours of structured exercise) in the last 3 months, 5) low alcohol consumers (< 2 drinks/day), 6) stable hypertension, diabetes, and hyperlipidemia and 7) non-frail (based on the criteria of Fried et al. (21)). It should be noted that 18 subjects had diabetes, hypertension and dyslipidemia at the same time, 13 had two of the above cited conditions and that 19 only had hypertension, 2 diabetes and 7 dyslipidemia. Subjects that meet any of the following criteria were excluded from the study: 1) milk protein intolerance or allergies, 2) subjects currently using natural health products such as N-acetylcysteine, α-lipoic acid supplements or dry whey protein supplements, 3) allergies or intolerance to soy, 4) major surgery in the year prior to testing, 5) acute coronary (e.g. myocardial infarction) or vascular event within the last year as well as uncontrolled coronary heart disease (e.g. progressive angina), 6) stroke within the past 2 years, 7) orthopedic limitations that limit the participation in the exercise training program, 8) uncontrolled thyroid or pituitary disease, 9) history of angioedema, 10) medication that has a major effect on cognitive function, 11) signs of early dementia as assessed by a the Mini-Mental State Examination (MMS < 24), 12) weight loss of more than 4 kg (or more than 5 % of body weight) over the last 6 months, 13) all medications that can interfere with muscle mass such as corticosteroids (e.g. prednisone), testosterone replacement or anabolic drugs such as Megace® and 14) subjects currently undergoing immunosuppressive therapy or hormone replacement therapy. All procedures in the protocol were approved by the Institut de Recherches Cliniques de Montréal (IRCM) Ethics Committee, which complies with the current laws of Canada. All participants were fully informed about the nature, goal, procedures and risks of the study, and gave their informed consent in writing.

Body composition

Body weight (BW), fat mass and lean body mass (LBM) were measured using dual energy X-ray absorptiometry (General Electric Lunar Prodigy; standard mode; software version 12.30.008, Madison, WI, USA). Standing height (± 0.1 cm) was measured using a wall stadiometer (Perspective Enterprises, Michigan, USA). Body mass index [BMI = Body weight (kg)/Height (m²)] was calculated.

Muscle strength

Lower body muscle strength was assessed using an Atlantis Precision leg press machine (Atlantis Inc. Laval, Canada). Subjects were aligned with the ball of their feet on the footplate of the machine at shoulder width so that their knee angle approximated 90° of flexion. Keeping their back flat against the chair, participants were assisted to the starting position with extension of their legs outward. A complete repetition consisted of flexing the knees to 90° and slowly returning to a complete extension of the legs stopping before full knee extension. Subjects were asked to start their session with a light walk on a treadmill for 10 min. Muscle strength was measured using a one-repetition maximum (1-RM) technique. The first set was used as a warm up of 10 repetitions with a light initial load set by the training supervisor. Thereafter, the load was increased until maximal effort was achieved. The 1-RM was typically determined within 5 trials with a 4 min rest between each trial. Failure was defined as a lift falling short of the full range of motion. If none of the trials yielded a 1-RM, the Wathan equation (22) was used to extrapolate the 1-RM. Two muscular strength indices were calculated by dividing the weight lifted (1-RM) in kg by body weight (kg) and by LBM (kg).

Protein supplementation

Each subject received either a daily dose of 20 g (2 x 10 g pouches) of the cysteine-rich whey protein isolate (Immunocal®) or a daily dose of 20 g (2 x 10 g pouches) of casein during a 135-day period. The whey protein isolate (Immunocal®) has been licensed as a natural health product in Canada and as a dietary supplement in the US. Casein is a milk protein fraction with similar caloric and nitrogen content to whey (23). Each subject was provided with a cup with the lid and instructions to take 2 pouches daily: one at breakfast and the second mid-morning or mid-afternoon. However, on the days that the subject performed their resistance training sessions, the daily 2 pouches were instructed as follows: one at breakfast and the second within 1 hour after the end of each exercise session. The subjects were instructed to mix the content of each pouch with a total of 8 to 12 oz. of their favorite juice or cool water in the cup provided by Immunotec Inc. (Vaudreuil, Quebec, Canada). The use of milk and hot liquids were avoided. The study compound was in powdered form. Participants were supplied with 90 pouches representing a 45-day supply. Subjects were provided with a refill of their treatment every 45 days. Participants returned the used and unused pouches at the IRCM after each 45-day supply. It should be noted that both the subjects and the research team at IRCM (principal investigators, study coordinators and trainers) were blinded as to which supplement was given until the end of the trial. Participants were instructed to maintain their usual eating habits.

Resistance training program

The resistance training program was performed on 3 non-consecutive days (i.e., Monday, Wednesday and Friday) during the entire 135-day treatment period. Each training session started with a warm-up of low intensity walking on a treadmill for 10 min. The resistance training program consisted of the following exercises: 1) leg press, 2) chest press, 3) lateral pull downs, 4) shoulder press, 5) arm curls, and 6) tricep extensions. These exercises provide a total body resistance training program for all of the major muscle groups of the body. The subjects were given a target load range and attempted to keep each set (n = 3) within the target range by adjusting the load to allow the prescribed number of repetitions (n = 10). Resting periods were 1-2 minutes between sets. The intensity of the exercise training sessions was approximately 80 % of the 1-RM. Each exercise session was individually monitored for optimal progression by qualified IRCM trainers.

Statistical analyses

The data are expressed as the mean ± standard deviation (SD). Unpaired t-tests were performed to compare means between groups at baseline and post intervention (day 135). A repeated measures ANOVA was used to examine changes following the intervention within each group and between groups (time x group interaction); the Bonferroni correction was applied. When a significant time × group interactions was found, a paired t-test analysis was performed to detect the time effect in each group. We also calculated the effect size (mean difference pre-post intervention / SD) for all variables that were significantly improved after the intervention. It should be noted that participants were included in the analysis if they completed at least 80 % of the exercise sessions and used 80 % of the study product. Statistical analysis was performed using SPSS 20 for Windows (Chicago, IL). Significance was set at a p value of 0.05 or less.

Results

A total of 99 participants were recruited in the study and 84 completed the 135 day study period. Of these, 17 participants were excluded from the analysis since they did not attend at least 80 % of their training sessions and used 80 % of the study product (7 in the Immunocal® group and 10 in the casein group). Fifteen other participants (8 in the Immunocal® group and 7 in the casein group) dropped out of the study. Subjects dropped out for the following reasons: health problems not related to training (n = 2), minor injury related to training (n = 3), did not like the training program (n = 2), did not tolerate the study product (n = 2), conflicting time schedules (n = 1), travel distance to the research unit (n = 1) and unspecified reasons (n = 4). Therefore, a total of 67 subjects (33 in the casein group and 34 in the Immunocal® group) were included in the analysis. Mean attendance for the exercise sessions in the participants that completed the study was 91 % in the casein group and 90 % in the Immunocal® group. In addition, mean use for the study product in the participants that completed the study was 96 % in the casein group and 97 % in the Immunocal® group.

Physical characteristics of the participants are presented in Table 1. No differences between groups were observed for all variables at baseline and post intervention. Significant increases in absolute muscle strength as well as muscle strength normalized by BW and LBM within both groups (p < 0.0001) were observed. Relative changes for all three muscle strength variables (absolute, normalized by BW and by LBM) were 31.0 %, 30.9 % and 30.0 %, respectively in the casein group as well as 39.3 %, 39.9 % and 43.3 %, respectively for Immunocal®, yielding between group differences favoring Immunocal® of 8.3 %, 9.0 % and 13.3 %, or approximately 10 % increase in these parameters. Significant group effects favoring Immunocal® versus casein for muscle strength expressed in kg/kg BW and in kg/kg LBM (p < 0.05) were noted. In addition, upper LBM was significantly increased in both groups after the intervention. No significant changes were found between pre-and-post intervention in both groups for body weight, BMI, fat mass, total LBM and lower LBM. Finally, the effect size for all three muscle strength variables (absolute, normalized by BW and by LBM) was 0.68, 0.79 and 0.92, respectively in the casein group as well as 1.1, 1.38 and 1.43, respectively in the Immunocal® group.

Table 1.

Physical characteristics of the participants before (baseline) and after the intervention (day 135)

Variables	Casein (n = 33)			Immunocal® (n = 34)
	Pre	Post	% Δ	Pre	Post	% Δ
Age (years)	71.0 ± 4.6	-	-	69.9 ± 3.6	-	-
Sex	♀ = 26, ♂ = 7	-	-	♀ = 26, ♂ = 8	-	-
Body weight (kg)	66.2 ± 9.2	66.4 ± 9.4	0.31	66.2 ± 9.9	65.9 ± 9.6	-0.34
Height (m)	1.61 ± 0.07	-	-	1.63 ± 0.08	-	-
Body mass index (kg/m2)	25.4 ± 2.8	25.4 ± 2.9	0.31	24.9 ± 2.8	24.8 ± 2.7	-0.34
Total lean body mass (kg)	39.9 ± 7.6	40.1 ± 7.2	0.59	40.1 ± 7.7	40.5 ± 7.3	1.04
Lower lean body mass (kg)	13.99 ± 2.69	14.05 ± 2.64	0.8	13.36 ± 3.67	14.05 ± 2.46	2.3
Upper lean body mass (kg)	4.29 ± 1.20	4.48 ± 1.21*	4.0	4.31 ± 1.07	4.46 ± 1.11*	3.6
Fat mass (kg)	24.2 ± 5.4	24.1 ± 5.7	-0.1	23.8 ± 6.1	23.3 ± 5.9	-1.67
Muscle strength (kg)	101.2 ± 34.9	129.2 ± 47.5*	31.0	102.9 ± 28.5	137.7 ± 34.4*	39.3
Muscle strength (kg/kg BW)	1.52 ± 0.45	1.94 ± 0.60*	30.9	1.56 ± 0.35	2.11 ± 0.44*	39.9†
Muscle strength (kg/kg LBM)	2.52 ± 0.65	3.20 ± 0.82*	30.0	2.53 ± 0.62	3.43 ± 0.63*	43.3†

Values are mean ± SD; BW: body weight; LBM: lean body mass; * Significantly different from baseline values (p < 0.0001); † A significant group effect (Casein vs. Immunocal®) in favor of the Immunocal® group was detected for muscle strength expressed in kg/kg BW and in kg/kg LBM (p < 0.05)

Discussion

We hypothesized that the cysteine-rich whey protein (Immunocal®) group would experience a greater increase in muscle strength and LBM versus the control group (casein) in elderly individuals. Our results show a significant increase in muscle strength (~10 % more) in subjects who were supplementing with Immunocal® versus casein. Furthermore, the effect size for all muscle strength indices in the Immunocal® group was higher than in the casein group. This could be encouraging for health professionals since impairments in muscle strength may be associated with a decrease in functional capacities, a lower walking speed, falls and disability (1, 24, 25). The present study results showing improvement in performance by the group taking Immunocal® are in line with the study of Lands et al. (18). Though in a young healthy population, Lands et al. (18) showed that supplementation with a cysteine-rich undenatured whey protein (Immunocal®) for 3 months significantly increased peak power and 30 sec work capacity in a whole leg isokinetic cycle test by ~13 % when compared to the control group (casein). In another cross sectional study, Benton et al. (26) observed that higher protein intake was positively associated with upper and lower body muscle strength in older individuals with chronic obstructive pulmonary disease.

Despite the differences in the increase in muscle strength, the intervention in both groups did not increase total LBM. In contrast, using an animal model and without resistance training, a recent study showed that long-term supplementation of a cysteine-based antioxidant could delay or prevent the loss of muscle mass during aging (27). Interestingly, there is evidence to suggest that changes in muscle strength appear to be independent from changes in muscle mass (24, 28, 29, 30). Hughes et al. (24) reported that age-related changes in muscle mass explained less than 5 % of the variance in muscle strength. Moreover, muscle strength, and not muscle mass, was shown to be associated with physical limitations (31) and mortality (32). Furthermore, muscle strength has been shown to be more correlated with functional capacities than muscle mass in the elderly (28, 33).

Due to their differences in absorption rates and bioavailability, several studies have compared the potential anabolic effect of whey vs. casein protein ingestion in combination with a resistance training program and have produced contrasting results (34, 35, 36, 37). For example, one study reported that whey protein supplementation (1.5 g/kg/day) with resistance training during 10 weeks had a greater increase in LBM and muscle strength than casein in recreational bodybuilders (35). In contrast, Demling et al. (36) showed that casein protein ingestion (1.5 g/kg/day) in combination with a hypocaloric diet plus resistance training for 12 weeks had greater gains in muscle strength than the whey protein group in overweight police officers. Finally, Wilborn et al. (37) observed no differences between whey and casein protein supplementation (24 g) in combination with a resistance training program during 8 weeks for LBM and muscle strength in collegiate female athletes.

Contradictory results have also been observed between studies examining the effect of a protein supplementation (none of which were a cysteine-rich whey protein) in combination with resistance training in the elderly (38, 39, 40, 41, 42). For example, Leenders et al. (40) reported no differences between groups for muscle strength and LBM after 24 weeks of resistance training with or without 15 g of milk protein per day in 60 healthy elderly subjects. Tieland et al. (41) showed similar increases in muscle strength between groups after 24 weeks of resistance training in 62 frail elderly individuals, however, the increase in LBM was superior in the group receiving 15 g of milk protein supplementation twice daily. Finally, Kim et al. (39) observed higher increases in muscle strength and leg muscle mass after 3 months of resistance training with or without 6 g of leucine-rich essential amino acids (which represents ~1.5 % by weight in whey protein (43)) per day in 77 sarcopenic elderly patients. A possible explanation for these contradictory results may be the dosage of protein intake. Interestingly, a recent study examined the dose-response effect of muscle protein synthesis with the ingesting of 4 different whey protein isolate dosages (0, 10, 20 and 40 g) after completing a resistance exercise in younger and older adults. Results show that a dose of at least 20 g of protein may be necessary for increasing muscle protein synthesis in older adults. However, muscle protein synthesis was additionally increased with a dose of 40 g of protein in the elderly subjects (44). Future interventional studies could examine the effects of different dosages of whey protein including cysteine-rich (20 vs. 40 g) on muscle strength and LBM in older adults.

It should be noted that our findings are limited to a selected population of sedentary non-frail elderly individuals who volunteered to participate in an exercise program 3 times per week, a daily nutritional intervention protocol and complied at least 80 % of the time. Furthermore, no measurement of energy and macronutrient intake was performed. Moreover, the present resistance training program targeted the upper body more. Conversely, our results are strengthened by using a double blind randomized controlled study for 135 days (4.5 months) in a relatively large sample size of well-characterized elderly individuals.

In conclusion, our findings showed increases in muscle strength in both groups, despite no increases in LBM, after resistance training, however, significant additional increases were observed in muscle strength with the addition of a cysteine-rich whey protein (Immunocal®) versus casein. Therefore, healthcare professionals could consider recommending the combination of resistance training and a cysteine-rich whey protein supplement (Immunocal®) when planning nutritional and exercise intervention programs. Further studies may want to investigate the long-term effect of cysteine-rich whey protein supplementation and in combination with resistance training on changes in muscle strength and/or LBM in the elderly, which could provide useful information on long term outcomes such as functional capacity, quality of life and mortality.

Acknowlegments: RRL is a Fonds de Recherches du Québec en Santé (FRQ-S) scholar and holds the J-A DeSève research chair. We thank Louis Coupal, Impacts inc. for the statistical analysis and Annie Karabadjian, Medscope Inc. for data monitoring. Immunotec Inc. provided funds for this study, supplied the study products and read the manuscript before submission. The sponsors had no role in data collection, data interpretation, or the writing of the manuscript. The study is registered (clinicaltrials.gov) under the identifier: NCT00935610.

Conflict of interests: Immunotec Inc. provided funds for this study.

Ethical standards: The IRCM ethics committee approved this study, which complies with the current laws of Canada.