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Year-long Changes in Protein Metabolism in Elderly Men and Women Supplemented With a Nutrition
Cocktail of β-Hydroxy-β-methylbutyrate (HMB), L-Arginine, and L-Lysine
Shawn Baier, Darcy Johannsen, Naji Abumrad, John A. Rathmacher, Steven Nissen and Paul Flakoll
JPEN J Parenter Enteral Nutr 2009 33: 71
DOI: 10.1177/0148607108322403

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Original Communication Journal of Parenteral and
Enteral Nutrition
Volume 33 Number 1

Year-long Changes in Protein Metabolism January/February 2009 71-82

© 2009 American Society for
Parenteral and Enteral Nutrition
in Elderly Men and Women Supplemented 10.1177/0148607108322403
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β-Hydroxy-ββ-methylbutyrate (HMB),
L-Arginine, and L-Lysine
Shawn Baier, MS1; Darcy Johannsen, PhD1; Naji Abumrad, MD, FACS 2;
John A. Rathmacher, PhD3,4; Steven Nissen, DVM, PhD3,4; and Paul Flakoll, PhD1
Financial disclosure: The study was supported by an NIH-NIA SBIR grant (R44AG20897) through Metabolic Technologies, Inc., Ames,
Iowa. Steven Nissen is the inventor of several patents related to HMB and is the CEO of Metabolic Technologies, Inc., Ames, Iowa, which
is the patent licensee of HMB. Paul Flakoll and Naji Abumrad were co-inventors on one of the HMB patents as well. John Rathmacher
is an employee at Metabolic Technologies, Inc.

Background: A major contributing factor to the loss of mobility HMB/Arg/Lys supplement, lean tissue increased over the year
in elderly people is the gradual and continuous loss of lean body of study while in the control group, lean tissue did not change.
mass. Objectives: To determine whether supplementation of an Compared with control, HMB/Arg/Lys increased body cell mass
amino acid cocktail daily for 1 year could improve the age- (BIA) by 1.6% (P = .002) and lean mass (DXA) by 1.2% (P =
associated changes in protein turnover and lean body mass in .05). The rates of protein turnover were significantly increased
elderly people. Design: Elderly (76 ± 1.6 years) women (n = 39) 8% and 12% in the HMB/Arg/Lys-supplemented group while
and men (n = 38) were recruited for a double-blinded controlled rates of protein turnover decreased 11% and 9% in the control-
study. Study participants were randomly assigned to either an supplemented subjects (P < .01), at 3 and 12 months, respec-
isonitrogenous control-supplement (n = 37) or a treatment- tively. Conclusions: Consumption of a simple amino acid-related
supplement (HMB/Arg/Lys) consisting of β-hydroxy-β-methyl- cocktail increased protein turnover and lean tissue in elderly
butyrate, L-arginine, and L-lysine (n = 40) for the 1-year study. individuals in a year-long study. (JPEN J Parenter Enteral Nutr.
Lean tissue mass was measured using both bioelectrical-impedance 2009;33:71-82)
analysis (BIA) and dual energy x-ray absorptiometry (DXA).
Rates of whole-body protein turnover were estimated using primed/ Keywords: L-arginine; L-lysine; β-hydroxy-β-methylbutyrate,
intermittent oral doses of 15N-glycine. Results: In subjects taking the aging; FFM; lean mass; protein turnover

A
fter the age of 40 years, lean body mass decreases in hormone activity.8 These factors contribute to the grad-
at a rate of approximately 8% per decade,1-4 and ual decrease in the rate of protein synthesis that is
this loss accelerates to approximately 15% per observed with elderly people.9-11 It is estimated that the
decade after the age of 70.3 The underlying causes of this rate of protein synthesis in humans decreases by as much
loss appear to be multifactorial5 and involve motor unit as 38% by middle age (50 years) and by 55% with
remodeling,6 age-related disease effects,7 and a decrease advanced age (70+ years) when compared with younger
individuals (∼20 years).11 Obviously, strategies aimed at
stemming the loss of muscle associated with these various
From the 1Department of Food Science and Human Nutrition, underlying pathologies should attempt to support or
Iowa State University, Ames; 2Department of Surgery, Vanderbilt increase the rates of protein synthesis.
University Medical Center, Nashville, Tennessee; 3Department of The implications of muscle loss in elderly people range
Animal Science, Iowa State University, Ames, IA; and 4Metabolic from the obvious relationship between lean body
Technologies, Iowa State University Research Park, Ames, IA. mass/strength and falling fractures12,13 to the less obvious
Received for publication June 6, 2007; accepted for publica- relationship between lean tissue loss and morbidity and
tion April 15, 2008. even mortality.14-16 Therefore, it is clear that decreasing the
Address correspondence to: Steven Nissen, DVM, PhD, rate of age-related muscle loss has the potential to
Department of Animal Science, 313 Kildee Hall, Iowa State markedly affect disease processes and also to improve qual-
University, Ames, IA 50011; e-mail: [email protected]. ity of life issues related to mobility and self-sufficiency.

71
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72 Journal of Parenteral and Enteral Nutrition / Vol. 33, No. 1, January/February 2009

To date, most attempts to reverse this age-associated if they were equal to age 65 years or greater and were
loss in protein have not directly addressed complex within the predetermined range for the functionality
changes in protein metabolism in elderly people and have score (“Get-up-and-Go” test,28 > 10 seconds but < 20 sec-
generally failed to improve lean tissue or function. We onds). Subjects were excluded if they were receiving
previously reported that daily supplementation of β- active treatment for liver or kidney diseases. They were
hydroxy-β-methylbutyrate (HMB), L-arginine (Arg), and also excluded for uncontrolled hypertension, uncon-
L-lysine (Lys) for 12 weeks positively altered measure- trolled diabetes, or classified as morbidly obese (body
ments of protein synthesis, and simultaneously increased fat > 30% for men and > 35% for women). All subjects
functionality, strength, and body composition in elderly were informed about the nature and risks of the experi-
women.17 This study was based on the premise that net mental procedures before each subject gave written
increases in protein balance should be the result of informed consent in accordance with and as approved by
improvements in the rates of both protein synthesis and the Institutional Review Board of Iowa State University.
protein breakdown, keeping in mind that both processes Figure 1 illustrates the subject screening process,
are inter-related; Assimon and Stein have shown that in randomization to treatments, and follow-up time points
most cases where protein synthesis is stimulated there is for testing over the course of the 12-month study.
a parallel increase in protein breakdown.18 We proposed Randomization was performed by 1 of the investigators
that adding L-arginine in this mixture could stimulate (J.A.R.) and the randomization scheme and codes
protein synthesis. Furthermore, we hypothesized that remained blinded to the investigators (S.B., D.J., and P.F.)
daily dietary supplementation with L-lysine (1.5 g), a lim- collecting and entering the data and in monthly contact
iting amino acid in elderly people, would ensure adequate with the subjects. The subjects underwent an initial
supplies to accommodate the increase in protein synthe- screening and were randomized to treatments. Testing
sis.19 HMB was added to the mixture as it has been shown consisted of an initial (0 months) and follow-up testing at
to slow protein breakdown and increase protein synthesis 3, 6, 9, and 12 months over the course of the 12-month
in tissues, especially muscle.20-22 Our previous prelimi- study. Of the 129 potential subjects recruited, 104 were
nary report17 only investigated the effects of the targeted randomized and assigned to either the HMB/Arg/Lys
nutrition support in women and, second, the study dura- treatment (n = 52) or the control treatment (n = 52).
tion was only for 3 months. This relatively short time of Ninety-five individuals (47 women, 76.4 ± 1.6 years; 48
measurement becomes important considering the years it men, 76.6 ± 1.3 years) completed initial testing, and 83
takes for muscle loss to occur. Lastly, protein turnover of those subjects completed the initial follow-up testing at
studies were only performed in a small subset of the sub- 3 months. During the year-long study, 8 control-treated
jects at the end of the study, thus precluding definitive subjects and 10 HMB/Arg/Lys-treated subjects were
conclusions on the mechanism of the effect. dropped from the study, leaving 77 subjects that com-
The current study determines whether a once-daily pleted the entire 12-month study. After the study, all sub-
cocktail of HMB, L-arginine, and L-lysine (HMB/Arg/Lys) jects were notified of their treatment group and asked if
over a 1-year period can improve, in both men and they were informed, purposely or inadvertently, of their
women, the age-associated changes in protein turnover treatment assignment before completion of their final
and ultimately lean body mass in the aged. We estimated testing. Of those subjects responding, it was the conclu-
body composition in all subjects using 2 independent sion of the authors that the subjects, as well as the study
methods, and we measured protein turnover in a subset coordinators in direct contact with the subjects, remained
of subjects both at the beginning and at the end of the blinded throughout the study.
year study. We hypothesized that lean tissue loss, which
occurs with aging, could be attenuated by nutritionally
targeting the underlying problems of protein synthesis Supplementation
and proteolysis with a combination of HMB/Arg/Lys.
Before the start of the study, subjects were randomly
assigned to 1 of the treatment groups (HMB/Arg/Lys or
Subjects and Methods control). During the course of the year-long study, sub-
jects and observers were blinded to the treatments. The
experimental group received an orange-flavored drink
Subjects
(HMB/Arg/Lys; Juven® Ready-to-Drink, Abbott Nutrition,
One hundred twenty-nine men and women were recruited Chicago, IL), which contained HMB (CaHMB, 2 or 3 g),
from 8 senior citizen centers and adult assisted-living and L-arginine (5 or 7.5 g), L-lysine (lysine HCl, 1.5 or 2.25 g),
care facilities in central Iowa. One week before the initi- and ascorbic acid (0.1 g). The dosage for each subject was
ation of the study, each subject filled out a questionnaire based on research showing that the HMB dosage of 38
detailing his/her medical history. Subjects were included mg per kg of body weight23 resulted in a greater increase

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 Nutrition Intervention in Elderly Patients / Baier et al 73

Inclusion Criteria Assessment (n = 129)

Men (n = 61) Women (n = 68)

Excluded or Declined
Participation (n = 25)

Randomized (n = 104)
Men (n = 50) Women (n = 54)

Allocated to Allocated to
HMB/Arg/Lys Treatment (n = 52) Control Treatment (n = 52)
Men (n = 25) Women (n = 27) Men (n = 25) Women (n = 27)

Declined or Unable to Declined or Unable to

Participate (n = 2) Participate (n = 7)

Completed Initial Baseline testing (n = 50) Completed Initial Baseline testing (n = 45)
Men (n = 25) Women (n = 25) Men (n = 23) Women (n = 22)

Dropped (n = 7) Dropped (n = 5)

3-Month Completed Follow-up (n = 43) Completed Follow-up (n = 40)

Men (n = 23) Women (n = 20) Men (n = 19) Women (n = 21)

Dropped (n = 1)

Completed Follow-up (n = 43) Completed Follow-up (n = 39)

6-Month
Men (n = 23) Women (n = 20) Men (n = 18) Women (n = 21)

Dropped (n = 3) Dropped (n = 2)

Completed Follow-up (n = 40)

9-Month Completed Follow-up (n = 37)
Men (n = 21) Women (n = 19)
Men (n = 17) Women (n = 20)

Completed Follow-up (n = 40) Completed Follow-up (n = 37)

12-Month Men (n = 21) Women (n = 19) Men (n = 17) Women (n = 20)

Figure 1. Subject screening, randomization, and follow-up flowchart. HMB/Arg/Lys, β-hydroxy-β-methylbutyrate, L-arginine,
and L-lysine.

in lean body mass and muscle strength compared with 76 group received a similar orange-tasting isonitrogenous
mg per kg of body weight.23 Subjects weighing 68 kg or and isocaloric drink consisting of a mixture of nonessen-
less were assigned a single dosage of 2 g of HMB, 5 g of tial amino acids (alanine 5.6 g, glutamic acid 0.9 g,
L-arginine, and 1.5 g of L-lysine, and those subjects glycine 3.1 g, serine 2.2 g and containing 0.1 g of ascor-
weighing > 68 kg were assigned a single dosage of 3 g of bic acid, and matched with the treatment based on net
HMB, 7.5 g of L-arginine, and 2.25 g of L-lysine. The control weight of the packet content (Table 1). Each dose for

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74 Journal of Parenteral and Enteral Nutrition / Vol. 33, No. 1, January/February 2009

Table 1. Basic Ingredient Formulations of Control

and β-hydroxy-β-methylbutyrate, L-argine, and L-lysine
(HMB/Arg/Lys) Treatments
Control HMB/Arg/Lys

Item ≤ 68 kg ≥ 68 kg ≤ 68 kg ≥ 68 kg
a
Amino Acids, g 11.8 17.7 6.5 9.8
Nitrogen, g 1.84 2.75 1.84 2.75
HMB, g 0 0 2 3
Calcium,b mg 270 405 270 405
Phosphorus, mg 100 150 100 150
Potassium, mg 260 390 260 390
Vitamin C, mg 100 150 100 150
Other, gc 11.2 16.8 16.5 24.7
a
Control: L-alanine, L-glutamate, glycine, and L-serine. Figure 2. Timeline for the 5-day diet record, 3-day urine
b
Calcium source is from CaHMB for HMB/Arg/GLN formulations; collection and 24-hour primed/intermittent oral doses of the
calcium source is from Gluconal Calcium for Control formulations. 15
N-glycine isotope study.
c
Maltodextrin, natural and artificial sweeteners, flavors, and colors.

either the HMB/Arg/Lys or the control treatment was studied. The average body weight for the control and
supplied in a separate white foil packet, and was allocated HMB/Arg/Lys treatment groups at 0 months was 74 ±
by subject number. The subjects were told to mix the sim- 2.8 and 77.4 ± 3.6 kg, respectively. Rates were estimated
ilar white powdered contents of the individual packets by using primed/intermittent oral doses of 15N-glycine
with approximately 235–350 mL of water depending on (98%, Cambridge Isotopes, Cambridge, MA) and meas-
packet/dosage size, and instructed to consume 1 packet urements of urinary 15N-urea and 15N-ammonia enrich-
daily in the morning with breakfast. ments. The steady state conditions for these methods
were previously described and validated by Flakoll et al,17
with steady state nitrogen flux attained from 9 to 18
Body Composition hours.17 Protein flux was calculated by a modified ver-
sion of the end product method of Picou and Taylor-
Body composition measurements were obtained at 0, 3, 6,
Roberts,25 by dividing mean 15N-urea and 15N-ammonia
9, and 12 months of supplementation using a single-fre-
enrichments for steady state time points (9–18 hours
quency bioelectrical impedance analyzer (BIA) at 50 kHz
post-isotopic infusion) by the rate of 15N-glycine delivery
and 800 AA (BIA Model 101, RJL Systems, Clinton
and multiplying by 6.25 to convert nitrogen to protein
Township, MI), and data were analyzed using the equa-
flux (turnover). Total (fecal and urine) nitrogen excre-
tions in “Cyprus,” version 2.5 (RJL Systems), which are
tion was multiplied by 6.25 and was subtracted from
based on the equations reported by Kotler et al.24 Dual
protein flux to obtain an estimate of daily rate of protein
energy x-ray absorptiometry (DXA; Hologic QDR Delphi)
synthesis. Total nitrogen excretion was estimated by
was used to measure lean mass of the upper and lower
multiplying urine nitrogen by 1.25 and was based on
extremities as well as the whole body. One trained opera-
direct measurements of urine and fecal nitrogen excre-
tor was responsible for conducting and analyzing the
tion in elderly women.26,27 During each of the collection
scans for all subjects. Baseline body composition and sub-
ject characteristics are shown in Table 2. and measurement periods, protein intake was estimated
over a 5-day period from dietary records with pre- and
postmeasurement interviews conducted by a study rep-
resentative. Subjects were instructed to keep the dietary
Protein Turnover Estimation
record of the 4 days before and the day of the tracer
Rates of whole-body protein flux, proteolysis, and pro- ingestion. The records were analyzed for single nutrient
tein synthesis were estimated at 0, 3, and 12 months of amounts and nutrient percentages and ratios for each
supplementation in a subset of patients from 5 of the 8 day as well as for the average of all 5 days (Nutritionist
senior citizen centers and adult assisted-living and care Pro Nutrition Analysis Software, Axxya Systems LLC,
facilities (Table 3). Twenty-two subjects (14 women and Stafford, TX). Protein intake was subtracted from protein
8 men) in the control group and 19 subjects (9 women flux to obtain a daily rate of proteolysis. Figure 2 illustrates
and 10 men) in the HMB/Arg/Lys treatment group were the timeline for the protein turnover study periods.

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 Nutrition Intervention in Elderly Patients / Baier et al 75

Table 2. Summary of Baseline Characteristics for Elderly Men and Women

Completed 12 months of Supplementation Treatment

Control (n = 37) HMB/Arg/Lys (n = 40)

Item Male (n = 17) Female (n = 20) Male (n = 21) Female (n = 19)

Lowa/high doseb 3/14 11/9 2/19 9/10

Age, yearsc 76.1 ± 1.6 76.2 ± 1.6 75.5 ± 1.4 75.3 ± 1.7
Weight, kg 79.9 ± 2.5 70.1 ± 2.7 86.8 ± 3.3 70.6 ± 2.7
Fat mass,d % 19.0 ± 1.1 39.5 ± 1.5 21.0 ± 1.0 39.2 ± 1.5
Fat-Free-Mass,d % 81.0 ± 1.1 60.5 ± 1.5 79.0 ± 1.0 60.8 ± 1.5

HMB/Arg/Lys, β-hydroxy-β-methylbutyrate, L-arginine, and L-lysine.

a
Low = subjects weighing < 68 kg: 2 g of HMB, 5 g of Arg and 1.5 g of Lys.
b
High = subjects weighing > 68 kg: 3 g of HMB, 7.5 g of Arg and 2.25 g of Lys.
c
Mean ± SE.
d
Values are bioelectrical impedance analysis.

Table 3. Baseline Characteristics of the Subset of Elderly Men and Women in the Protein Turnover Studies
Treatment

Control (n = 22) HMB/Arg/Lys (n = 19)

Item Male (n = 8) Female (n = 14) Male (n = 10) Female (n = 8)

a b
Low /high dose 2/6 7/7 1/9 5/4
Age, yearsc 82.3 ± 2.1 78.6 ± 1.8 79.5 ± 2.0 79.0 ± 2.6
Weight, kg 79.9 ± 5.1 70.6 ± 3.1 83.1 ± 4.8 71.0 ± 4.7
Fat mass,d % 19.2 ± 2.4 41.7 ± 1.5 20.2 ± 1.5 40.1 ± 2.2
Fat-Free-Mass,d % 80.9 ± 2.4 58.3 ± 1.5 79.8 ± 1.5 59.9 ± 2.2

HMB/Arg/Lys, β-hydroxy-β-methylbutyrate, L-arginine, and L-lysine.

a
Low = subjects weighing < 68 kg: 2 g of HMB, 5 g of Arg and 1.5 g of Lys.
b
High = subjects weighing > 68 kg: 3 g of HMB, 7.5 g of Arg and 2.25 g of Lys.
c
Mean ± SE.
d
Values are bioelectrical impedance analysis.

Strength and Functionality reported.29,30 A quality of life questionnaire (SF-36 Health

Survey)31 and a psychological profile32 were also adminis-
Strength was measured in the upper and lower extremi- tered at 0, 3, 6, 9, and 12 months. These questionnaires
ties using an isokinetic system for knee extension (Biodex, were primarily administered as a means to evaluate the
System 3 Quickset, Shirley, NY) and an isometric subject’s perception of quality of life or mood.
dynamometer for hand grip strength (Grip Track, Jtech
Medical, Heber City, UT). Functionality was tested at 0,
3, 6, 9, and 12 months of supplementation using the Blood and Urine Collection/Analysis
“Get-up-and-Go” and “Get-up” performance tests.28
At 0, 3, 6, 9, and 12 months, blood samples were taken
from a superficial arm vein into Vacutainers (Becton
Questionnaires Dickinson, Vacutainer Systems, Rutherford, NJ) after an
overnight fast. Urine samples were also taken at this time.
Each subject was asked to fill out an adverse events ques- The blood and urine samples for each collection time
tionnaire before starting the study and again monthly point were processed and placed in a specimen delivery
thereafter. This questionnaire was used to monitor any kit and shipped to Labcorp, Inc (Kansas City, MO) at 4°C
changes in indicators of health and well-being as previously for analysis. Whole blood was collected for a complete

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76 Journal of Parenteral and Enteral Nutrition / Vol. 33, No. 1, January/February 2009

blood cell count with a differential white blood cell count

and platelet count. Serum was collected and used for a A BIA Fat-Free-Mass
1.2
comprehensive metabolic panel (Chem12) with 7 addi- Control HMB/ARG/LYS
tional blood chemistries and a lipid profile. Urinalysis 1

Change, kg
with gross examination was performed on the urine. An 0.8
aliquot of urine and serum was obtained and stored at 0.6
−20°C for HMB measurement to assess compliance in 0.4
taking the supplements. 0.2
0
0 3 6 9 12
Months
Compliance
B BIA Body-Cell-Mass
Monthly compliance questionnaires and daily supple- 1.2
mentation records were given during the study with sub- 1
Control HMB/ARG/LYS

jects in both groups, indicating that compliance was

Change, kg
0.8
approximately 95% over the entire 12-month study. Urine 0.6
HMB levels were measured to assess compliance33 and in
0.4
the HMB/Arg/Lys-supplemented subjects averaged 364 ±
0.2
71 nmol/mL, significantly greater than those in the
control-supplemented subjects, which was 25 ± 2 nmol/mL 0
0 3 6 9 12
(P < .05). Months
C DXA Lean Mass
1.2
Statistics 1
Control HMB/ARG/LYS
Change, kg

The primary outcome of this study was the attenuation of 0.8

lean body mass loss in an older adult population in a year- 0.6
long study. A power analysis (NCSS Statistical Software, 0.4
Kaysville, UT) was completed before the initiation of the 0.2
study using lean body mass data from Flakoll et al.17 For the
power analysis calculation, a 0.7 kg increase in lean mass 0 3 6 9 12
was anticipated for the HMB/Arg/Lys treatment group dur- Months
ing the year-long study, whereas no change in lean mass was
Figure 3a, 3b, 3c. Graphic illustration of body composition
expected to occur in the control treatment group. The cal-
data for elderly men and women during 12 months of supple-
culation determined that a total sample size of 80 subjects,
mentation with HMB, L-arginine, and L-lysine. A description of
40 on each treatment, would be needed to detect a differ- the statistical design was outlined in the Methods section. A,
ence in lean mass over the 12-month study. The calculation Bioelectrical impedance analysis (BIA) fat-free mass (FFM)
assumed a 25% dropout rate over the duration of the study. showed that HMB/Arg/Lys resulted in a significant linear
Mean ± SEM was calculated for each variable and for increase in FFM (P = .002). B, BIA Body Cell Mass (BCM)
changes in each variable over the 12-month period. The showed HMB/Arg/Lys resulted in a significant linear increase in
mixed models procedure of the Statistical Analysis System BCM (P = .001). C, Dual energy x-ray absorptiometry (DXA)
for Windows (Release 8.02, SAS Institute, Cary, NC) was Lean showed HMB/Arg/Lys resulted in a significant quadratic
used to analyze the data. Body composition, protein increase in DXA lean mass (P = .05). Data points are means ±
turnover, strength, and functionality data were analyzed SEM and the lines are a fit to the data.
by repeated measure ANOVA that included the starting
value as a covariate and the main effects of site, gender, at multiple points (0, 3, 6, 9, and 12 months) were eval-
and treatment. There were no gender by treatment inter- uated with both linear (Time x Treatment) and quadratic
actions. We anticipated that elderly people would respond (Time x Time x Treatment) treatment contrasts. Only
to nutritional supplementation in either a linear or quad- those subjects completing 12 months of study were
ratic manner. Linear and quadratic responses were deter- included in the model and reported here (n = 37 control
mined because long-term evaluation of independent main and n = 40 HMB/Arg/Lys); these results were presented
effect variables may not always give a best fit to the linear in Figures 3 and 4 and Tables 4 and 5. Intent-to-treat
response. This is because the initial rate of response may analysis was performed and included data from the 83
not be sustained at the same rate during the entire study subjects that completed at least the 3-month follow-up.
period. Therefore, the main effects of the nutritional The intent-to-treat statistical analyses of body composition
cocktail on protein turnover and lean body mass over time and protein metabolism were not different from the

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 Table 4. Effect of Long-Term Supplementation of β-hydroxy-β-methylbutyrate (HMB), L-Arginine (Arg),
and L-Lysine (Lys) on Body Composition in Elderly Men and Women
Control HMB/Arg/Lys
Treatment
Change (months) Change (months) contrasts, P

Item 0a 3 6 9 12 SEM 0 3 6 9 12 SEM Linear Quadratic

Weight, kg 74.6 ± 2.1 0.29 0.28 0.63 −0.07 0.34 79.1 ± 2.5 0.37 0.51 1.61 0.67 0.37 .02 .87
BIA
Fat Free Mass, kg 52.2 ± 2.1 0.22 0.01 0.14 0.12 0.22 55.8 ± 2.3 0.48 0.48 1.00 0.88 0.25 .002 .42
Body Cell Mass, kg 22.7 ± 1.0 0.17 0.14 0.04 0.02 0.18 24 ± 1.1 0.43 0.64 0.64 0.58 0.19 .001 .25
Fat mass, kg 22.1 ± 1.7 0.25 0.32 0.54 −0.11 0.27 23.3 ± 1.5 −0.10 0.03 0.61 −0.18 0.32 .61 .64
Total body water, L 38.8 ± 1.3 0.20 0.06 0.17 0.13 0.20 41.4 ± 1.6 0.41 0.42 0.89 0.81 0.22 .002 .58
Intracellular water, L 20.7 ± 0.9 0.16 0.13 0.040 0.027 0.16 22.5 ± 1.0 0.24 0.42 0.43 0.38 0.15 .01 .65
Extracellular water, L 18.1 ± 0.5 0.03 −0.07 0.11 0.11 0.14 19.4 ± 0.6 0.11 −0.06 0.43 0.38 0.17 .16 .90
DXA
Lean Mass, kg 46.4 ± 1.6 0.20 0.10 0.18 0.17 0.18 49.2 ± 1.7 0.73 0.56 0.81 0.55 0.23 .18 .05
Fat mass, kg 24.1 ± 1.3 0.05 0.50 0.24 0.20 0.26 25.1 ± 1.2 −0.41 0.16 0.44 0.61 0.26 .07 .09
Bone mineral content
Total body, g 2417 ± 99.8 12.4 18.2 23 14 10.5 2491 ± 93.9 4.1 6.4 14.3 9.2 10.2 .62 .40
Bone mineral density, g/cm2 1.042 ± 0.032 −0.001 −0.007 −0.003 −0.003 0.005 1.065 ± 0.036 0.001 −0.005 −0.009 −0.002 0.007 .63 .98

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BLA, dioelectrical impedance analysis; DXA, dual energy x-ray analysis.
a
Basal data are expressed as uncorrected mean ± SEM. The changes from basal based on uncorrected means are expressed as a mean ± pooled SE.

77
78 Journal of Parenteral and Enteral Nutrition / Vol. 33, No. 1, January/February 2009

Table 5. Protein Turnover in Elderly Men and Women After 3 and 12 Months of
Supplementation With β-hydroxy-β-methylbutyrate (HMB), L-Arginine (Arg), and L-Lysine (Lys)
Control (n = 22) HMB/Arg/Lys (n = 19)
Treatment
Month Month contrasts P =

0a 3 12 0 3 12 Linear Quadratic

Urinary nitrogen g/kg/d 0.13 ± 0.01 0.14 ± 0.01 0.15 ± 0.01 0.13 ± 0.01 0.15 ± 0.01 0.14 ± 0.01 .79 .69
Nitrogen balanceb g/kg/d 0.00± 0.005 −0.03 ± 0.005 −0.03 ± 0.006 0.001 ± 0.006 −0.02 ± 0.006 −0.01 ± 0.006 .24 .59
Protein intakec g/kg/d 0.99 ± 0.02 0.96 ± 0.02 0.98 ± 0.03 0.98 ± 0.03 1.01 ± 0.03 1.04 ± 0.03 .23 .25
Protein turnoverd g/kg/d 5.3 ± 0.2 4.7 ± 0.2 4.9 ± 0.2 5.3 ± 0.2 5.8 ± 0.2 6.1 ± 0.2 .007 .02
Protein synthesis g/kg/d 4.3 ± 0.2 3.6 ± 0.2 3.8 ± 0.2 4.4 ± 0.2 4.7 ± 0.2 5.0 ± 0.2 .007 .03
Protein breakdown g/kg/d 4.3 ± 0.2 3.8 ± 0.2 3.9 ± 0.2 4.4 ± 0.2 4.8 ± 0.2 5.1 ± 0.2 .01 .04
Protein balancee g/kg/d −0.01 ± 0.03 −0.17 ± 0.03 −0.17 ± 0.04 0.00 ± 0.04 −0.12 ± 0.04 −0.09 ± 0.04 .24 .59

a
LSMean ± SEM.
b
Nitrogen Balance = Nitrogen intake – (Urinary nitrogen x 1.25).
c
Protein intake was estimated over a 5-day period from dietary records.
d
Protein turnover is the calculated protein flux.
e
Protein Balance = Protein synthesis – Protein breakdown.

analysis on the complete 12-month analyses and, there- supplementation for 1 year resulted in a significant linear
fore, only the 12-month data were reported. increase (P = .002) in FFM as compared to the control
Adverse events questionnaires were analyzed as cate- supplement elderly men and women. Furthermore, sub-
gorical data and the main effect of treatment was deter- jects taking the HMB/Arg/Lys supplement had a 0.58 ±
mined using the Cochran-Mantel-Haenszel test.34 0.19 kg increase in body cell mass (BIA), while body cell
Statistical significance was set at P < .05 for all analyses. mass in the control-supplemented group was unchanged
0.02 ± 0.18 kg (Figure 3B); the starting time 0 values for
Results control and HMB/Arg/Lys were 22.7 ± 1 and 24.0 ± 1.1
kg, respectively. After 12-months of supplementation, the
Subjects HMB/Arg/Lys treatment response was significantly
increased in a linear manner (P = .001).
Table 2 summarizes the data for the subjects’ treatment Similar to the FFM data from BIA, DXA measures of
assignment, age, height, body weight, and composition. A lean mass in those subjects taking the HMB/Arg/Lys
total of 77 subjects completed the 12-month study with supplement showed a 0.55 ± 0.23 kg increase after the
21 of the HMB/Arg/Lys-supplemented men and 17 of the year-long study, while the increase in lean mass for the con-
control-supplemented men finishing the study, while 19 trol-supplemented group was not different from zero, 0.17
of the HMB/Arg/Lys-supplemented women and 20 of the ± 0.18 kg (Table 4). The HMB/Arg/Lys-supplemented older
control-supplemented women completed the final 12- adults resulted in a significant maintenance in DXA lean
month follow-up testing. The subjects that dropped from over the 12 month study as compared to the control-
the study did so because of the demanding commitment supplemented older adults (Figure 3C; quadratic contrast,
of a year-long study and not due to any adverse events. P = .05). The DXA time 0 data for control and HMB/Arg/Lys
were 46.4 ± 1.6 and 49.2 ± 1.7 kg, respectively.
While both BIA and DXA showed increases in lean
Body Composition
mass, there were no significant changes in fat mass or
Data from both BIA and DXA show HMB/Arg/Lys- percent body fat with either BIA or DXA measures
supplemented subjects increased fat-free mass (FFM) (Table 4). However, there was a significant linear (P = .002)
and/or lean mass compared with the control-supple- increase in total body water in the HMB/Arg/Lys-
mented subjects (Figure 3; Table 4). FFM time 0 data for supplemented older adults compared with the control
control and HMB/Arg/Lys were 52.2 ± 2.1 and 55.8 ± 2.3 group. The increase in total body water was accompanied
kg, respectively. Subjects taking the HMB/Arg/Lys sup- by a significant linear increase (P = .01) in the intracellu-
plement had a 0.88 ± 0.25 kg increase in FFM (BIA), lar water compartment without a significant change in the
while FFM in the control-supplemented group was not extracellular water compartment of the HMB/Arg/Lys-
changed, 0.12 ± 0.22 kg (Figure 3A). The HMB/Arg/Lys supplemented older adults during the 12-month study.

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 Nutrition Intervention in Elderly Patients / Baier et al 79

Functionality and Strength

Protein turnover
Measures of functionality and strength were not differ- 1.5
ent among treatment groups. However, there was a

Change, g . kg –1 . d–1
gradual loss of handgrip and leg strength in both the 1 *
control-supplemented and HMB/Arg/Lys-supplemented
*
0.5
groups over the 12-month study. Measures of the
“Get-up-and-Go” and “Get-up” functionality tests remained 0
unchanged during the study and were not different –0.5
between treatments.
–1
3 Months 12 Months
Estimation of Daily Whole-Body Control HMB/ARG/LYS
Protein Turnover
Rates of protein turnover were approximately 20% greater Figure 4. The change in protein turnover in elderly men and
in the HMB/Arg/Lys-supplemented group compared with women after 3 and 12 months of supplementation with HMB,
the control-supplemented subjects at 3 months and 12 L-arginine, and L-lysine. Mean changes are shown with SEM indi-
months of supplementation (Table 5). At 3 and 12 cated by the bars. *HMB, L-arginine, and L-lysine showed a sig-
nificant change in protein turnover at 3 and 12 months (P ≤ .01).
months, the rates of protein turnover were significantly
increased 8% and 12%, respectively, in the HMB/Arg/Lys-
Discussion
supplemented group while rates of protein turnover
decreased 11% and 9%, respectively, in the control-
The current study demonstrates that a nutritional supple-
supplemented subjects (P < .01; Figure 4). At 3 months,
ment containing the amino acid-related compound
the rate of protein synthesis was increased by 7% in the
HMB in combination with L-arginine and L-lysine
HMB/Arg/Lys-supplemented group (from 4.4 ± 0.2 to 4.7 ±
(HMB/Arg/Lys) resulted in an increase in whole-body
0.2 g/kg/d), while the rate of protein synthesis decreased
protein turnover and an increase in lean body mass in eld-
by 18% in the control-supplemented subjects (from 4.3 ±
erly men and women over a 1-year period compared with
0.2 to 3.6 ± 0.2 g/kg/d, P < .01, Table 5). The changes in
no change in lean mass in a similar group of men and
protein synthesis reported at 3 months were also observed
women receiving an isonitrogenous control supplement.
at 12 months (Table 5), where the rate of protein synthesis
These results extend the findings from an earlier study
increased by 14% in the HMB/Arg/Lys-supplemented
showing that the same cocktail of amino acids (HMB/Arg/
group (5 ± 0.2 g/kg/d), while the rate remained lower in
Lys) was effective in enhancing lean body mass in elderly
the control-supplemented subjects (3.8 ± 0.2 g/kg/d,
women over a 3-month period.17 A similar strategy using
P < .01, Table 5). However, because of similar changes in
HMB/ARG/glutamine (GLN) has also been used to mini-
the rates of protein breakdown, protein balance remained
mize AIDS-related muscle loss35 and cancer-related
the same for both treatment groups. Five-day recall
cachexia.36 Thus, the idea of nutritionally targeting the
dietary records indicated that protein and caloric intakes
components of the protein turnover rate, namely protein
were not different between treatment groups (data not
synthesis and proteolysis, would appear to be useful tool
shown).
in stemming the loss of muscle in a variety of conditions.
Previous studies with protein/calorie supplementa-
Estimates of Health and Well-being tion have failed to maintain or reverse age-related losses
in lean mass in elderly.37-40 In contrast, the current nutri-
Mood and indicators of psychological well-being were not tional cocktail treatment of HMB/Arg/Lys and testos-
different between the treatment groups, and there was no terone treatment41,42 are, to date, the only 2 interventions
change in either group over the 12-month study. Evaluation shown to affect elderly lean body mass loss (without exer-
of the SF-36 questionnaire suggested no improvement in cise). Both these interventions showed a significant lean
quality of life for the HMB/Arg/Lys-supplemented group body mass increase, but both failed to induce any treat-
compared with the control-supplemented group. ment effects on strength and functionality. Although the
exact reasons for this disconnect between lean body mass
and strength are not known, the similarity of effects on
Safety and Adverse Events
increased lean body mass could suggest a common mech-
There were no significant changes over time on blood or anism such as enhancements of protein synthesis.
urine markers of hepatic or renal function, blood lipids, In the current study, the use of the amino acid cock-
or chemistry either within or between treatment groups. tail was effective in increasing lean tissue and appeared to

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80 Journal of Parenteral and Enteral Nutrition / Vol. 33, No. 1, January/February 2009

be related to an increase in protein turnover and/or pro- hematology, and physical measures were not altered during
tein synthesis, although net protein balance failed to the study. Additionally, relatively simple and safe supple-
reach significance. The increase in protein synthesis in mentation protocol in the current study stands in contrast
the HMB/Arg/Lys-supplemented group is consistent with to many previous studies using ineffective protein/calorie
the known affects of L-arginine to stimulate protein supplementations,37-40 or interventions such as exer-
synthesis.43,44 In addition, L-lysine supplementation cise40,57,58 or hormone therapy,41,42 which are only applica-
could also allow protein synthesis to increase in an L- ble to a very small percentage of elderly individuals.
lysine-limiting situation as L-lysine is considered to be the Further studies determining the impact of preventing mus-
most limiting amino acid in muscle protein synthesis.19,45 cle loss on fractures, quality of life issues, and ultimately
Lastly, recent studies in mice have also shown HMB to the impact on the healthcare costs would seem warranted.
stimulate protein synthesis46 by similar mechanisms as In conclusion, once-daily consumption of a simple
those known for leucine.46-48 amino acid-related cocktail increased protein turnover
Protein breakdown was also increased in the and lean tissue in elderly people in a year-long study.
HMB/Arg/Lys group matching the rise in the rates of
protein synthesis. These changes in protein breakdown
contrast with many studies showing that HMB supple- Acknowledgments
mentation alone resulted in reduced protein breakdown
(as measured by the urinary 3-methyl-histindine method) The authors dedicate this work to the memory of Dr. Paul
and consequently increasing lean body mass in humans Flakoll who passed away at the conclusion of this study. In
undergoing resistance exercise.49 The mechanism of addition to being a colleague to each of us, he was role
HMB-induced reduction in muscle proteolysis appears to model for the ideals we aspire to as scientists, teachers, and
be related to increased gene expression of the ubiquitin- as friends and family. He is missed by all who knew him.
proteosome pathway.21,50 In the current study, the failure The authors thank the volunteers for their faithful partici-
to measure a relative decrease in protein breakdown and pation in this study. We also thank the students and staff of
a change in protein balance (and nitrogen balance) con- the Center for Designing Foods to Improve Nutrition and
trasts with the significant increases in FFM and body cell the staffs of the Eastern Star Masonic Home, Boone, IA;
mass. The failure to measure a statistical effect on net Iowa Veterans Home, Marshalltown, IA; Green Hills
protein balance could be due to variation in urine collec- Retirement Community, Ames, IA; Northcrest Community,
tions, isotope measures, inaccurate diet recall, or the Ames, IA; Bethany Life, Story City, IA; United Church of
reduced power of a smaller subset of subjects used in the Christ, Ames, IA; Heartland Senior Services Center, Ames,
turnover studies. In addition, the failure to show a change IA; and Iowa State University Retirees, Ames, IA.
in net protein balance may be due to inadequate protein Shawn Baier was involved in the study design, subject
intakes, as Kortebein et al recently demonstrated that recruitment, data collection, analysis, and writing of the
elderly subjects with protein intakes of 0.8 g/kg/d were manuscript. Darcy Johannsen was involved in subject
in negative nitrogen balance before a bed rest study.51 recruitment and data collection. Naji Abumrad was
However, the subjects in our study had protein intakes of involved in study design and review and editing of the man-
1 g/kg/d from the diet plus 0.2 g/kg/d from the supple- uscript. John Rathmacher was involved in study design,
ments, and these intakes should have adequately met data collection, statistical analysis, and writing of the man-
their requirement. Consequently, we cannot define the uscript. Steven Nissen was involved in study design, data
exact mechanism whereby lean body mass increased in interpretation, and writing of the manuscript. Paul Flakoll
the HMB/Arg/Lys group. was involved in study design and data collection.
Aging is associated with a reduction in whole body
protein turnover and a progressive decline in the body’s
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