Protein Supplementation at Breakfast and Lunch for 24 Weeks beyond Habitual Intakes Increases Whole-Body Lean Tissue Mass in Healthy Older Adults 1 3

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1 The Journal of Nutrition Nutrient Requirements and Optimal Nutrition Protein Supplementation at Breakfast and Lunch for 24 Weeks beyond Habitual Intakes Increases Whole-Body Lean Tissue Mass in Healthy Older Adults 1 3 Catherine Norton, 4,5 Clodagh Toomey, 4,5 William G McCormack, 4,5 Peter Francis, 4,5 Jean Saunders, 6 Emmet Kerin, 4 and Philip Jakeman 4,5 * 4 Human Science Research Unit, Center for Interventions in Inflammation, Infection, and Immunity, 5 Food for Health Ireland, and 6 Statistical Consultancy Unit, University of Limerick, Limerick, Ireland Abstract Background: Key areas of research on the preservation of lean tissue mass (LTM) during aging are determinations of the protein requirement and optimal protein intake at meals. Objective: The aim of this study was to determine the effect of protein supplementation at breakfast and lunch for 24 wk beyond habitual intakes on whole-body LTM in healthy adults aged y. Methods: In a single-blinded, randomized, controlled design, 60 healthy older men and women (aged y) with a body mass index (in kg/m 2 ) of consumed either g/kg body mass of a milk-based protein matrix (PRO) or an isoenergetic, nonnitrogenous maltodextrin control (CON) at breakfast and midday meals, the lower protein containing meals of the day, for 24 wk. Dual-energy X-ray absorptiometry was used to measure the change in LTM. Results: After the intervention, protein intake in the PRO group increased from to g/kg for breakfast and from to g/kg for the midday meal. In response, LTM increased by 0.45 (95% CI: 0.06, 0.83) kg in the PRO group compared with a decrease of 0.16 (95% CI: 20.49, 0.17) kg in the CON group (P = 0.006). Appendicular LTM accounted for the majority of the difference in LTM, increasing by 0.27 (95% CI: 0.05, 0.48) kg in the PRO group compared with no change in the CON group (P = 0.002). Conclusions: Protein supplementation at breakfast and lunch for 24 wk in healthy older adults resulted in a positive (+0.6 kg) difference in LTM compared with an isoenergetic, nonnitrogenous maltodextrin control. These observations suggest that an optimized and balanced distribution of meal protein intakes could be beneficial in the preservation of lean tissue mass in the elderly. This trial was registered at clinicaltrials.gov as NCT JNutr2016;146:65 9. Keywords: Introduction healthy aging, sarcopenia, lean tissue mass, meal level protein intake, milk protein matrix The postmaturation decline in lean tissue mass (LTM) 7 is an insidious process with an estimated loss rate of 1 2%/y from the age of 50 y (1 4). Principal to the preservation of LTM is the maintenance of skeletal muscle mass for metabolic health and to 1 Supported by Food for Health Ireland and Enterprise Ireland grant CC Author disclosures: C Norton, C Toomey, P Francis, J Saunders, E Kerin, and P Jakeman, no conflicts of interest. WG McCormack was an employee of Carbery Ingredients on secondment to Food for Health Ireland during the in vivo data collection and analysis. 3 Supplemental Tables 1 4 are available from the Online Supporting Material link in the online posting of the article and from the same link in the online table of contents at 7 Abbreviations used: ALTM, appendicular lean tissue mass; BM, body mass; CON, maltodextrin control; LTM, lean tissue mass; MPS, muscle protein synthesis; PRO, milk-based protein matrix. * To whom correspondence should be addressed. phil.jakeman@ul.ie. defer the onset of sarcopenia and frailty. A number of underlying mechanisms contribute; however, to date, the main nutritional focus in relation to the preservation of LTM in aging has been protein requirement (5 10) and the age-specific change in muscle protein synthesis (MPS) in response to protein and amino acid intake (11 16). The current RDA for protein in healthy men and nonpregnant adult women of all ages is 0.8 g kg 21. d 21 of high-quality (i.e., high in essential amino acids) protein. On the basis of nitrogen-balance studies, the RDA for protein is nominally 2 SDs above the Estimated Average Requirement (0.66 g kg 21. d 21 ) and reflects the average minimal amount of protein (nitrogen) intake to balance nitrogen excretion and prevent a progressive loss of body protein (17). However, achieving nitrogen balance may be an inadequate outcome with respect to the age-related decline of LTM. Recent consensus statements suggest that a protein intake above the ã 2016 American Society for Nutrition. Manuscript received June 19, Initial review completed July 30, Revision accepted October 19, First published online November 18, 2015; doi: /jn

2 RDA (i.e., between 1.0 and 1.5 g kg 21. d 21 ) may be of benefit to the preservation of LTM in healthy adults (9, 10). Guideline recommendations for daily protein intake make no reference to meal-level protein intake or the optimal distribution of protein intake throughout the day. Estimates of meal-level dietary protein intake in the United States (17) and Europe (7) find the lower protein containing meal (normally breakfast, ;8 15 g) to be suboptimal with respect to stimulation of MPS, with only the higher protein containing meal (normally dinner, ;30 40 g protein) reaching a protein intake that is optimal for postprandial MPS (9, 14). Working within the current recommended guidelines for daily protein intake but redistributing the meal-level intake of protein across breakfast, lunch, and dinner may offer a strategy to optimize net protein balance. In support of this strategy, isoenergetic and isonitrogenous diets consumed evenly (;30 g protein/meal) throughout the day resulted in a 25% greater 24-h mixed-muscle protein fractional synthesis rate compared with a skewed (;10, 20, and 60 g protein/meal) daily protein intake (18). Independent of the daily amount, the source of protein intake can modulate the rate of MPS. Milk protein consists of ;80% casein and ;20% whey. Casein and whey are considered to be of high quality due to their high relative essential amino acid composition, rate of digestion and absorption, and bioactivity, which can be enhanced by partial hydrolysis (19, 20). The protein synthetic response to postprandial hyperaminoacidemia (21) and other bioactive stimuli provided by protein ingestion (20) is impaired in the skeletal muscle of older adults (12), particularly after a low (<30 g/meal) protein intake (5, 9) or when the dose of essential amino acids, leucine in particular, is low (11 14, 22). These findings suggest that a higher amount of protein intake per meal, ;0.40 g/kg, would be optimal for skeletal muscle MPS and to retain LTM in older adults. FIGURE 1 Participant flow chart. CON, maltodextrin control; PRO, milk-based protein matrix; $, women; #, men. To test this hypothesis, the present study sought to provide a high-quality protein supplement for 24 wk beyond habitual intakes to healthy, independent-living older adults. Supplemental protein, equivalent to 0.33 g protein/kg per day, was consumed in 2 equal parts with the lower protein containing meals of the day (i.e., breakfast and lunch). The primary outcome was the change in LTM measured by DXA. Methods Subject recruitment and progression. All of the participants were informed of the purpose of the study and all known risks before providing written, informed consent. The study was approved by the Faculty of Education and Health Sciences Research Ethics Committee (EHSREC10/45), University of Limerick. All procedures were carried out in accordance with the ethical standards outlined in the most recent version of the Declaration of Helsinki. The trial was registered at clinicaltrials.gov as NCT Figure 1 shows a flowchart of the progress from recruitment through completion at 24 wk. A convenience sample of healthy men and women (aged y) from the University of LimerickÕs Body Composition Study were invited by or word-of-mouth to participate in the study. After written, informed consent 178 volunteers were assessed for eligibility, submitted to a clinical examination, and provided a dietary record to a qualified dietitian. Those defined as healthy, free-living, fully mobile, and independent-living and with no indication of lactose intolerance or adverse reaction to dairy-based foods were invited to participate. Twenty subjects were excluded after screening (musculoskeletal issues: n = 6; medication: n = 6; celiac disease: n = 4; impaired fasting blood glucose concentration: n = 3; cancer in the previous 2 y, n = 1; and cardiopulmonary abnormality: n = 1) and 18 declined to participate further. Participants were then randomly assigned to receive a supplement containing either a milk-based protein matrix (PRO) or an isoenergetic, 66 Norton et al.

3 TABLE 1 Energy and macronutrient intakes in older adults in the CON and PRO groups at baseline and during the 24-wk intervention 1 CON PRO Baseline Intervention Baseline Intervention Energy, kcal/d Carbohydrate, %TEI Fat, %TEI Protein, %TEI Protein, g d Protein, g kg 21. d Values are means 6 SDs; n = 29 (24 women and 5 men; CON group) and n =31(22 women and 9 men; PRO group). CON, maltodextrin control; PRO, milk protein matrix; %TEI, percentage of total energy intake excluding alcohol. nonnitrogenous maltodextrin control (CON). A total of 60 of the 123 participants (49%) completed the study to 24 wk, 31 in the PRO group (22 women, 9 men) and 29 in the CON group (24 women, 5 men). Reasons for noncompletion of the study by group are provided in Figure 1. Study design. This was a randomized, single-blinded, controlled trial with time (baseline compared with 24 wk) and treatment group (CON compared with PRO) as the independent factors. Simple randomization was used to assign participants to a treatment group (CON or PRO). Supplements were prescribed relative to the median 4 levels of participantsõ body mass (BM; i.e., kg, median of 52.5 kg; kg, median of 67.5 kg; kg, median of 82.5 kg and kg, median of 97.5 kg). Each supplement dose, which provided g protein/kg of median BM or an isoenergetic amount of maltodextrin, was individually packaged in a foil sachet, and participants were instructed to reconstitute with water and consume twice daily with the lower protein meals of the day, normally breakfast and lunch. Five flavors were available to off-set flavor fatigue and to improve compliance. A mixed excess number of supplement portions were provided every 4 wk with instructions that all used or unused sachets must be returned. Subjects were blinded to the supplement composition assigned to them. Compliance was monitored by count-back at the end of each month. Body composition, measured by DXA, was performed at baseline and upon completion at 24 wk. Milk protein matrix. The energy and nutrient composition of the milk protein matrix per 100 g powder and per sachet is provided in Supplemental Table 1. The milk protein matrix was composed of a 9:2:1 ratio of milk protein concentrate [80% (wt:wt protein); Glanbia Nutritionals]; whey protein concentrate hydrolysate, degree of hydrolysis 32% [WPC DH % (wt:wt protein); Carbery Ingredients; and whey protein isolate hydrolysate, degree of hydrolysis 45% [WPI DH 45, 75% (wt:wt protein); Glanbia Nutritionals]. The total protein concentration was 72.7 g/100 g powder. The protein matrix was supplemented with 2187 mg/100 g powder of milk-based calcium (Trucal; Glanbia Nutritionals) and 57.3 mg/100 g powder cholecalciferol. On the basis of 2 sachets/d, the daily supplement of milk protein matrix, milk-based calcium, and cholcalciferol was 0.33 g/kg, 10 mg/kg, and 0.26 mg/kg median BM, respectively. Dietary intake. An estimate of participantsõ dietary intake was undertaken within a 14-d period before commencement of supplement intake. After comprehensive instruction and mentoring by a qualified dietitian, participants recorded food and fluid intake for 4 consecutive days (2 weekdays and 2 weekend days) by using an estimated food intake record. The dietitian provided oral and written instruction on recording food type, quantity, cooking method, and meal time. During follow-up and subsequent visits, the dietitian checked the diary for completeness and obtained any additional information required to improve the accuracy of the dietary record. Food intake data were coded and subsequently analyzed by proprietary software (WISP V4; Tinuviel Software). Modifications were made to the food-composition database to include recipes of composite dishes, nutritional supplements, generic Irish foods that were commonly consumed, and new foods on the market. All previous modifications to the food-composition database were also checked and updated from current manufacturersõ information as necessary. To remove some of the bias introduced by misreporting of food records, underreporters [i.e., reported energy intake to basal metabolic rate ratio of <1.1 (23)] were excluded from further analysis. Meal-level analysis of protein intake and amino acids was extracted for breakfast, midday (lunch), evening (dinner), and snack consumption. Clinical biochemistry. Blood samples were obtained by venipuncture of an antecubital vein. Serum and plasma were separated by centrifugation at 10,000 3 g at 4 C for 5 min and frozen at 278 C until analysis. Plasma insulin (catalog no ) and serum total 25-hydroxyvitamin D (catalog no ) were measured on a Roche Cobas e411 autoanalyzer (Roche Diagnostics). Plasma glucose (catalog no ) and serum calcium (catalog no. A28937), albumin (catalog no ), urea (catalog no ), cholesterol (catalog no ), TGs (catalog no ), HDL cholesterol (catalog no ), LDL cholesterol (catalog no ), and creatinine (catalog no. A60298) were measured on a UniCel DxC 800 autoanalyzer (Beckman Coulter). The interassay CV was <5% for all analytes other than vitamin D, total cholesterol (6.9%), and LDL cholesterol (6.0%). Anthropometric and body-composition measurements. Height was measured to the nearest 0.1 cm by using a stadiometer (Seca) and BM was measured to the nearest 0.1 kg (MC-180MA; Tanita UK Ltd.). Whole-body compositional analysis was performed using DXA as per the recommendations of the International Society of Clinical Densitometry (24). A Lunar idxa scanner with encore version 14.1 software (GE Healthcare) was used to capture and analyze whole-body scans. Calibration by means of a phantom block was performed daily. To standardize test conditions and tissue hydration, participants were instructed to refrain from strenuous exercise in the 12-h period before testing and to attend after an overnight fast. Participants consumed 500 ml water 1 h before the scan and were instructed to void and defecate, if required, immediately before measurement. The encore software provided first-pass segmentation of the total body into arm, leg, and trunk regions of interest. A qualified DXA technician checked for correct anatomic alignment and made manual adjustments where necessary. Whole-body and staturenormalized (kg/m 2 ) indexes of body fat and LTM were determined. The precision (root mean square CV) for repeated measurement of LTM was 0.55% and 1.2% for body fat mass. Statistical analysis. Baseline body-composition and clinical biochemistry data were checked for normality of distribution by using the Shapiro- Wilk test and expressed as means 6 SDs for normally distributed variables and medians (IQRs) for skewed variables. An independent t test or Mann- Whitney U test was used to compare differences between groups at baseline. The treatment effect was calculated as the change in DXAderived body composition from baseline to 24 wk and presented as means (95% CIs). These data were found to be normally distributed and analyzed by univariate ANOVA with treatment (CON compared with PRO group) and sex (men compared with women) as fixed factors. To determine the influence of baseline BM and LTM on the outcome, data were analyzed by ANCOVA with group and sex as fixed factors and baseline value of the dependent variable (LTM or BM) as the covariate. Dietary intake data are reported as means 6 SDs. Statistical analysis was performed by using PASW Statistics 18.0 for Windows (SPSS, Inc.). Significance (2-tailed) was set at P < 0.05 for all analyses. Results The data presented relate to the 60 healthy older adults (31 in the PRO and 29 in the CON group) who completed the study to week 24. Clinical biochemistry confirmed the general health of the subjects (Supplemental Table 2), which did not change in response to the intervention at week 24. Analysis of the 4-d estimate of dietary intake is provided in Table 1. There was nothing remarkable in the dietary intake and no difference was observed in the percentage of energy intake of Protein intake and lean tissue in older adults 67

4 carbohydrate, fat, or protein between CON and PRO groups. Intakes corresponded to current Institute of MedicineÕs recommendations (25) and were comparable to mean daily intakes from the Irish National Adult Nutrition Survey (23). Ninetyfour percent of subjects reported a dietary protein intake of $0.8 g kg 21. d 21, and 50% achieved a protein intake of $1.2 g kg 21. d 21. Breakfast and midday meals were identified as the lower protein containing (Supplemental Table 3) and lower leucine-containing (Supplemental Table 4) meals of the day. Supplementation in the PRO group achieved a balance of ;30% of daily protein (>0.4 g/kg) intake per meal and increased energy intake by 112 kcal/d for the PRO group and by 117 kcal/d for the CON group (Table 1). Anthropometric and body-composition values did not differ between CON and PRO groups at baseline (Table 2). The appendicular LTM (ALTM) index was characterized by reference to young adults of the same sex and ethnic group (26). All participants exceeded the ALTM index criterion value for age-related sarcopenia (27). The net change in BM and composition after the intervention was calculated by subtracting the baseline value from the value at week 24. The net change in BM (ΔBM) was positive in the PRO group and negative in the CON group, resulting in a difference in ΔBM between the groups of 0.9 kg at week 24 (Table 3). Similar to ΔBM, the net change in LTM (ΔLTM) was positive in the PRO group and negative in the CON group. The difference in ΔLTM between the groups was 0.6 kg at week 24. ΔLTM was greater in men than in women (treatment 3 sex interaction, P = 0.013). Covariate analysis confirmed that neither LTM nor BM at baseline exerted a significant influence on the change in LTM. No difference in the net change in body fat mass between the groups was observed. Discussion The study hypothesis was developed from recent evidence indicating that g high-quality protein is required per meal to optimally stimulate lean tissue protein synthesis (8, 14, 28) and that an even distribution of protein intake throughout the day may act as effective countermeasures to the age-related loss of LTM (29). To our knowledge the present study is the first longitudinal, randomized, controlled trial to test the validity of this hypothesis in healthy older adults. As measured by a 4-d estimate of food intake, the elderly participants in this study were considered protein sufficient TABLE 2 Anthropometric and body-composition values for healthy older adults in the CON and PRO groups at baseline 1 CON PRO P 2 Age, y Height, m 1.66 (1.61, 1.68) 1.63 (1.57, 1.68) 0.22 Body mass, kg BMI, kg/m Body fat, % Body fat mass, kg Lean tissue mass, kg 40.2 (36.6, 43.8) 40.1 (31.5, 48.8) 0.50 Lean tissue mass index, kg/m (14.0, 16.1) 14.9 (13.1, 16.6) 0.83 Appendicular lean tissue mass, kg 17.9 (16.2, 19.6) 17.7 (13.3, 22.1) 0.53 Appendicular lean tissue mass index, kg/m (6.1, 7.2) 7.0 (6.0, 8.0) Values are means 6 SDs or medians (IQRs); n = 29 (24 women and 5 men; CON group) and n = 31 (22 women and 9 men; PRO group). No significant differences were observed between groups. CON, maltodextrin control; PRO, milk protein matrix. 2 P values for the difference between PRO and CON groups analyzed by independent t test or Mann-Whitney U test. 68 Norton et al. TABLE 3 Change in body mass and composition in older adults in the CON and PRO groups from baseline to week 24 1 Treatment effect Treatment effect kg P 2 1-b % P 2 1-b BM CON (20.79, 0.35) (20.98, 0.64) 0.06 PRO 0.70 (0.01, 0.35) 0.95 (0.04, 1.86) BFM CON (20.59, 0.47) (21.48, 3.48) 0.56 PRO 0.24 (20.27, 0.75) 1.69 (20.41, 3.78) LTM CON (20.49, 0.17) (21.09, 0.49) PRO 0.45 (0.06, 0.83) 0.91 (0.03, 1.78) ALTM CON (20.15, 0.17) (20.77, 0.90) PRO 0.27 (0.05, 0.48) 1.11 (0.13, 2.10) 1 Data are means (95% CIs); n = 29 (24 women and 5 men; CON group) and n =31(22 women and 9 men; PRO group). Treatment effect = (value at week 24 value at baseline). ALTM, appendicular lean tissue mass; BFM, body fat mass; BM, body mass; CON, maltodextrin control; LTM, lean tissue mass; PRO, milk protein matrix; 1-b, statistical power. 2 P values for the difference in treatment effect, PRO compared with CON, analyzed by univariate ANOVA with treatment group and sex as fixed factors. (9, 10). Protein supplementation at breakfast and lunch for 24 wk beyond habitual intakes in these participants may not, therefore, appear to be warranted. However, consistent with meal-level protein intakes in the elderly (7, 17), lower protein intakes were evident at breakfast (;15 g) and midday meals (;20 g), with the majority of protein intake occurring at the evening meal (;40 g). Protein intake was, therefore, considered suboptimal for postprandial protein synthesis for 2 of the 3 main meals of the day. To effect the required change in meal-level protein intake we chose to provide a high-quality protein supplement to the lower protein containing meals of the day rather than attempt a redistribution of habitual intakes. ParticipantsÕ compliance and subjective rating of the supplementation regimen lend support to this approach. However, it was not possible to confirm that dietary supplementation had been attained without compensatory adjustment of dietary intakes. As a result of the intervention, protein intakes in the PRO group increased to $0.4 g/kg per meal. This amount of protein is considered within the range (62 SDs) of the optimal protein intake ( g/kg) to stimulate MPS in the elderly (22). Leucine intakes at breakfast and midday and evening meals increased to 1.8, 1.9, and 1.5 g, respectively, and were closer to the 3 g required to maximal stimulate MPS in older adults (29). These data support an augmented, postprandial rate of protein synthesis (30) in the PRO group compared with the CON group. It is difficult to accurately predict the change in BM that would result from a 6-mo increase in energy intake of ;100 kcal/d, accumulating to 16.8 Mcal at week 24. The measured outcome was a net increase in BM of 0.7 kg (;1%) in the PRO group compared with a net decrease of kg in the CON group (Table 3). Assuming a metabolizable energy density of ;7.7 kcal/kg for subjects with ;25 kg body fat (31), the BM equivalent to the increased energy intake would be ;2.2 kg. That this did not occur suggests that participants modified their habitual intake and/or energy expenditure over the course of the intervention, and to a greater extent in the CON than in the PRO group. Body-composition analysis revealed no difference in net body fat mass but a significant increase in net lean tissue mass (ΔLTM) in the PRO compared with the CON group. The difference in

5 ΔLTM between the groups was 0.61 kg at week 24. In the PRO group, the net increase in LTM accounted for approximately two-thirds of the net increase in BM. Because the majority of the net increase in LTM was located in the appendages (ALTM), skeletal muscle was probably the major beneficiary of the optimized protein intake in the PRO group. A recent systematic review assessed the effectiveness of nutritional supplementation on muscle mass in older people (32). The review analyzed 17 studies that included 1287 patients aged between 65 and 85 y. For inclusion in the analysis the period of supplementation had to be for $8 wk. Despite variable dietary regimens, compliance, and comparative controls, the review concluded that nutritional supplementation was proven to be effective in increasing LTM in elderly subjects. The present study overcame many of the limitations in study design identified in the review and supports its principal outcome. 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