Nutr (2001) Creatine, HMB and Hypertrophy

APPLIED NUTRITIONAL INVESTIGATION

Creatine and b-Hydroxy-b-Methylbutyrate (HMB)Additively Increase Lean Body Mass and Muscle

Strength During a Weight-Training ProgramEwa Jowko, MS, Piotr Ostaszewski, DVM, PhD, Michal Jank, DVM, Jaroslaw Sacharuk, PhD,

Agnieszka Zieniewicz, MS, Jacek Wilczak, PhD, and Steve Nissen, DVM, PhDFrom the Institute of Sport and Physical Education, Biala Podlaska, Academy of PhysicalEducation, Warsaw, Poland; the Department of Physiology, Biochemistry, Pharmacology

and Toxicology and the Department of Human Nutrition and Home Economics,Warsaw Agricultural University, Warsaw, Poland; and the Iowa State University,

Ames, Iowa, USA

We investigated whether creatine (CR) andb-hydroxy-b-methylbutyrate (HMB) act by similar ordifferent mechanisms to increase lean body mass (LBM) and strength in humans undergoing progressiveresistance-exercise training. In this double-blind, 3-wk study, subjects (n 5 40) were randomized toplacebo (PL;n 5 10), CR (20.0 g of CR/d for 7 d followed by 10.0 g of CR/d for 14 d;n 5 11), HMB(3.0 g of HMB/d;n 5 9), or CR-and-HMB (CR/HMB;n 5 10) treatment groups. Over 3 wk, all subjectsgained LBM, which was assessed by bioelectrical impedance analysis. The CR, HMB and CR/HMBgroups gained 0.92, 0.39, and 1.54 kg of LBM, respectively, over the placebo group, with a significanteffect with CR supplementation (main effectP 5 0.05) and a trend with HMB supplementation (maineffect P 5 0.08). These effects were additive because there was no interaction between CR and HMB(CR 3 HMB main effectP 5 0.73). Across all exercises, HMB, CR, and CR/HMB supplementationcaused accumulative strength increases of 37.5, 39.1, and 51.9 kg, respectively, above the placebo group.The exercise-induced rise in serum creatine phosphokinase was markedly suppressed with HMB supple-mentation (main effectP 5 0.01). However, CR supplementation antagonized the HMB effects on serumcreatine phosphokinase (CR3 HMB interactive effectP 5 0.04). Urine urea nitrogen and plasma ureawere not affected by CR supplementation, but both decreased with HMB supplementation (HMB effectP , 0.05), suggesting a nitrogen-sparing effect. In summary, CR and HMB can increase LBM andstrength, and the effects are additive. Although not definitive, these results suggest that CR and HMB actby different mechanisms.Nutrition 2001;17:558–566. ©Elsevier Science Inc. 2001

KEY WORDS: body composition, bioelectrical impedance, creatine, creatine phosphokinase,b-hydroxy-b-methylbutyrate, leucine, muscle

INTRODUCTION

Creatine (CR) andb-hydroxy-b-methylbutyrate (HMB) are sup-plements used to enhance the effects of weight training on musclemass. Although several mechanistic explanations have been of-fered to show how these compounds enhance exercise-responsivemuscle growth, to date there has been no experimental evidenceshowing whether any of these hypotheses are valid. One approachto this problem is to determine whether the effects of HMB and CRare independent. Additivity of those effects would support distinctmechanisms of action, and lack of additivity would support acommon mechanism. Thus, we investigated whether HMB and CRwere additive in relation to lean body mass (LBM) and strengthgains and whether these indicated independent mechanisms.

CR supplementation has been shown to raise muscle CR con-

centrations by approximately 15%,1–9 and that rise has been asso-ciated with increased LBM and strength.1,5,10,11It has been pos-tulated that the increase in muscle CR increases LBM byosmotically drawing more water into the muscle cell.1,4,10,11Thiscellular swelling would then stimulate protein synthesis in themuscle cells.1 As yet there have been no studies directly measuringmuscle-cell water changes in humans. In addition, in vitro studieshave suggested that CR can increase myosin synthesis but does notaffect total protein synthesis.12–14

The leucine metabolite, HMB, also has been shown to increaseLBM and strength in exercising humans.10,15–17Two mechanismsfor HMB action have been proposed. The first is based on theobservation that HMB can slow or suppress muscle proteolysisthat is elevated during exercise.15,17 The second possible mecha-nism concerns HMB being a precursor of muscle-cell cholesterol.The increase in muscle hypertrophy during weight training mightresult in a local deficiency in cholesterol in the muscle cell, whichmight result in inadequate cholesterol for membrane synthesis andthus slower cell growth or suboptimally functioning cell mem-branes due to a shortage of membrane cholesterol. Suppling crit-ical amounts of cholesterol precursor in muscle would allow themuscle to maintain and synthesize new muscle plasma membranes.This idea is supported by observations showing that muscle-

This work was supported by grant 5 PO6K 024 10 from the Polish StateCommittee of Scientific Research.

Correspondence to: Steven Nissen, DVM, PhD, Nutritional Physiology,313 Kildee Hall, Iowa State University, Ames, IA 50010. E-mail:[email protected]

Date accepted: January 19, 2001.

Nutrition 17:558–566, 2001 0899-9007/01/$20.00©Elsevier Science Inc., 2001. Printed in the United States. All rights reserved. PII S0899-9007(01)00540-8

membrane damage (leakage) was markedly decreased when HMBwas supplemented in the diet.15–18

Proposed mechanisms for CR and HMB action are clearlyspeculative at this time. However, if it can be shown that the effectsof HMB and CR are additive relative to LBM increase, this wouldprovide preliminary evidence consistant with the proposed mech-anisms of action. Conversely, if the effects of HMB and CR are notadditive, then one of the two proposed mechanisms would bewrong. We also examined several metabolic markers of cell me-tabolism, nitrogen metabolism, and strength to support the LBMgain data. Together these data should establish similarities ordifferences between CR and HMB mechanisms of action.

MATERIALS AND METHODS

Subjects

Forty healthy male subjects 19 to 23 y old were enrolled into thestudy. All had been involved in prior exercise but none were highlytrained. Potential subjects were excluded from the study if they hadevidence or history of any disease, recent joint or bone injury, orobesity. Subjects also were excluded if they had participated inresistance-exercise programs in the previous 6 mo or had con-sumed HMB or CR in the previous 3 mo before the study. Allsubjects were screened by blood analysis, urinalysis, physicalexamination, and body composition. The study was approved bythe Ethical Committee of the Academy of Physical Education inWarsaw, and informed written consent was obtained from allsubjects before the beginning of the study.

Dietary Control

All subjects were asked to consume three meals per day in thestudents’ cafeteria. Each week the subjects completed a question-naire detailing all food consumed during three non-consecutive24-h periods (one fell on a weekend day).3 The data were used tocalculate dietary intakes for each participant by using the Dietuscomputer program based on European dietary norms.

Experimental Groups

Table I summarizes the experimental periods, measurements, andcollections made during the study. All subjects were randomized totreatment groups in a double-blind fashion, and LBM was mea-sured. In the placebo group, one subject dropped out during thefirst week, leaving 10 subjects.

CR was given as creatine-monohydrate in powder form (99%creatine-monohydrate; Sigma Aldrich, Milwaukee, WI). Each sub-ject was instructed to mix the powder in 250 mL of orange juice.HMB was supplied in capsules containing 0.25 g of CaHMB(Metabolic Technologies, Ames, IA, USA). Each four-capsuleserving supplied 1.0 g of CaHMB, and each subject took theplacebo or HMB capsules three times per day. Placebo (rice flour)capsules were identical in look and taste to the HMB capsules andwere taken as the HMB capsules. Glucose (Sigma Aldrich) wasused as the powder placebo and was mixed in orange juice, as wasthe CR.

The placebo group consumed 20 g of glucose and 3 g ofplacebo capsules for the first 7 d and 10 g of glucose plus theplacebo capsules for the remainder of the study. The CR grouptook 20 g of CR/d (loading dose) for the first 7 d and 10 g/dthereafter (maintenance dose) plus placebo capsules. The HMBgroup took 20 g of glucose and 3 g of HMB per day for thefirst7 d and 10 g of glucose plus 3 g of HMB for theremainder of thestudy. The CR/HMB group took 20 g of CR and 3 g of HMBdailyfor the first 7 d and then 10 g of CR and 3 g of HMB daily for theremainder of the study.

Progressive Weight-Training Regimen

Each subject was tested for a one-repetition maximum (1-RM)before the study in all of the exercises except the sit-up crunch.The number of lifts was minimized during testing so as not toconstitute a training effect. Weight training continued three timesper week throughout the study with at least 1 d of rest betweensessions. The strength-training program consisted of concentricand eccentric isotonic lifting exercises that worked each musclegroup with free weights. The selection of exercises was based on

TABLE I.

EXPERIMENTAL DESIGN AND SAMPLING SCHEDULE

Baseline Week 1 Week 2 Week 3

TT W R F S U M T W R F S U M T W R F S U M T W R F S U M

Body composition* X XBlood sample† X X XPreliminary exercise‡ X XResistance exercise X X X X X X X X X XUrine collection§ X X XDietary-recall questionnaire X X X X X X X X XPlacebo, CR, HMB, and CR/HMB

supplementationX X X X X X X X X X X X X X X X X X X X X

* Bioelectrical impedance analysis.† Blood was collected between 7:00 and 8:00AM after an overnight fast.‡ Subjects were tested for maximum lifting capacity (1-RM) during the screening period.§ Twenty-four–hour collections were from 7:00AM on Wednesday to 7:00AM on Thursday during screening and week 2 and from 7:00AM on Mondayto 7:00AM on Tuesday at the end of the treatment period.CR, creatine; CR/HMB, supplementation with creatine andb-hydroxy-b-methylbutyrate; HMB, calciumb-hydroxy-b-methylbutyrate; T, W, R, F, S, U,M, Tuesday, Wednesday, Thursday, Friday, Saturday, Sunday, Monday, respectively; X, days when this protocol was followed or administered.

Nutrition Volume 17, Numbers 7/8, 2001 559Creatine and HMB Aid Muscle Growth

the training program for beginners; the seven exercises werechosen to involve the main large muscle groups as follows: chest,upper back, shoulders, arms, abdominal, and legs. The trainingprogram used is shown in Table II.

The initial weight lifted was calculated by using 1-RM as100%. The training weight was then estimated at the level at whichthe subject could make 5 to 15 repetitions before failure. Theweight lifted was increased slowly to keep the number of repeti-tions and sets the same. For example, if the subject easily com-

pleted or exceeded the planned number of repetitions in the last set,then the following calculation was used to estimate an increase inweight to be lifted during the next session. The number of plannedrepetitions was subtracted from the number of completed repeti-tions and divided by two. The value obtained was the number ofkilograms, which were then added to the existing weight to havethe target weight to be lifted at the next session. Each session wasmonitored by trained supervisors who recorded the weights liftedand repetitions performed.

TABLE II.

TRAINING PROGRAM FOR SUBJECTS RECEIVING CREATINE,b-HYDROXY-b-METHYLBUTYRATE, A COMBINATION OF THESE,OR PLACEBO

Muscle group Type of exercise

Weight lifted in % 1-RM

Total repetitionsWarm-up Main exercise

Chest Bench press 40

151;

50

121;

60

101*

702 75

83

61

Upper back Power clean 35

121

45 2 50

153

57

Shoulders, arms, upper back Behind-the-neck press 40

151;

50

121;

60

101

70

83;

75

61

61

Biceps Biceps curls 40

151;

50

121;

60

101

702 75

6 2 83

61

Triceps Triceps extensions 40

101;

50

81

60 2 65

64

42

Legs Back squat 50

81;

60

61

70

54

34

Abdominal Sit-up crunch 15–203 5†

* (% 1-RM/n repetitions)3 n sets.† n repetitions3 n sets.1-RM, one-repetition maximum

TABLE III.

PROTEIN, FAT, AND ENERGY INTAKES OF SUBJECTS SUPPLEMENTED WITH PLACEBO (n 5 10), CR (n 5 11), HMB (n 5 9), ORCR/HMB (n 5 10)

Treatment* P†

SEM‡Placebo CR HMB CR/HMB CR HMB CR3 HMB

Protein intake (g/d)Week 1 104.1 103.1 111.3 101.9 0.38 0.68 0.50 6.15Week 2 106.3 94.6 104.9 95.9 0.08 0.98 0.82 5.81Week 3 110.6 96.5 108.8 102.5 0.04 0.61 0.43 4.83

Fat intake (g/d)Week 1 110.8 107.2 108.4 105.9 0.62 0.76 0.93 5.84Week 2 110.3 105.5 115.6 103.6 0.12 0.80 0.50 5.34Week 3 120.6 115.0 122.4 121.7 0.63 0.52 0.73 6.97

Caloric intake (MJ/d)Week 1 12.81 12.51 13.16 12.54 0.39 0.75 0.76 0.54Week 2 13.97 12.37 14.67 12.19 0.001 0.72 0.47 0.59Week 3 13.41 12.19 13.40 12.47 0.07 0.80 0.80 0.58

* Values are least square means of placebo, CR, HMB, and CR/HMB treatments.† P values for treatment effects were determined with a 23 2 factorial analysis of variance.‡ Pooled standard error of the mean.CR, creatine; CR3 HMB, interaction between creatine andb-hydroxy-b-methylbutyrate; CR/HMB, creatine plusb-hydroxy-b-methylbutyrate; HMB,b-hydroxy-b-methylbutyrate

560 Jowko et al. Nutrition Volume 17, Numbers 7/8, 2001

Exercises were conducted after a warm-up at 40% to 60%(bench press, behind-the-neck press, bicep curl), 50% to 60%(back squat), or 40% to 50% (tricep extension) of the subjects’1-RM, followed by the main exercise at 70% to 75% (bench press,behind-the-neck press, bicep curl), 70% (back squat), or 60% to65% (tricep extension) of the 1-RM for each muscle group (TableII). The subjects took a break of approximately 3 to 4 min betweensets. To prevent any risk of contusion of the upper back muscles,the exercise for that group of muscles was conducted at only 45%to 50% of 1-RM. To evaluate the strength gains obtained duringthe exercise program, the 1-RMs were compared at the beginningand end of the study.

Blood and Urine Samples

Blood samples were collected during the screening period, duringweek 2, and at the end of the treatment period. Blood was takenfrom a superficial forearm vein after an overnight fast. Serum wascollected from the blood sample and then stored at226°C untilanalyzed for creatinine, creatine phosphokinase (CPK), and ureanitrogen (Alpha Diagnostics/Trace, San Antonio, TX, USA).Twenty-four–hour total urine collections were made during thescreening period, during week 2, and at the end of the treatment

period to measure urinary creatinine and urea nitrogen (AlphaDiagnostics/Trace).

Body Composition

Body cell mass (BCM), LBM, fat mass, total body water (TBW),and intracellular water (ICW) were assessed from reactance andresistance measurements with bioelectrical impedance analysis(single frequency of 50 kHz, hand held, four electrodes; RJL BIA101/S Body Impedance Analyzer, Akern, Clinton Township,MI).18–20 Measurements were collected in the morning after anovernight fast at the beginning and end of the treatment period.Subjects were to refrain from any physical activity for 12 h,withhold all liquids for 4 h, and urinate 30 min before the mea-surements. Electrodes were placed on the right hand and right footaccording to the manufacturer’s guidelines. Subjects were supine,with their arms 30 degrees away from their bodies, and they wereinstructed to keep their legs apart. Subjects were supine for ap-proximately 5 min, with the test performed during minute 5. Thebody weight, age, sex, and height for each subject were recorded.Reactance and resistance measurements were then processed withthe Akern Body Impedance Analyzer.6

TABLE IV.

AGE, HEIGHT, BODY WEIGHT, AND COMPOSITION OF SUBJECTS SUPPLEMENTED WITH PLACEBO, CR, HMB, OR CR/HMB BEFOREAND AFTER 3 WK OF EXERCISE

Treatment* P†

Placebo CR HMB CR/HMB CR HMB CR3 HMB SEM‡

n 10 11 9 10 — — — —Age (y) 20.4 21.4 20.8 21.2 0.07 0.81 0.48 0.38Height (cm) 177.0 179.2 180.0 180.0 0.50 0.29 0.50 0.67Body weight (kg)

Screen 73.77 75.72 75.06 73.48 0.92 0.83 0.50 2.59Week 3 74.77 77.73 76.40 76.88 0.48 0.89 0.62 2.48Difference 1.00 2.01 1.34 3.40 0.0001 0.007 0.10 0.31

Body fat (kg)Screen 11.34 12.63 12.13 11.05 0.88 0.69 0.30 1.12Week 3 11.50 12.83 12.21 12.04 0.56 0.94 0.48 1.03Difference 0.17 0.19 0.08 1.00 0.06 0.11 0.06 0.23

LBM (kg)Screen 62.43 63.09 62.93 62.43 0.95 0.95 0.71 1.58Week 3 63.27 64.89 64.18 64.82 0.46 0.80 0.76 1.56Difference 0.85 1.77 1.24 2.39 0.0005 0.08 0.73 0.27

BCM (kg)Screen 31.39 31.24 31.64 31.33 0.78 0.84 0.92 0.80Week 3 31.93 32.47 32.39 33.06 0.50 0.55 0.94 0.88Difference 0.54 1.23 0.75 1.73 0.01 0.26 0.65 0.32

TBW (L)Screen 44.65 44.99 44.91 44.59 0.98 0.95 0.80 1.28Week 3 45.41 46.64 46.03 46.66 0.46 0.81 0.82 1.28Difference 0.76 1.65 1.12 2.07 0.001 0.14 0.91 0.26

ICW (L)Screen 28.58 28.45 28.80 28.53 0.79 0.84 0.93 0.64Week 3 29.06 29.55 29.50 30.12 0.52 0.51 0.91 0.81Difference 0.48 1.10 0.70 1.59 0.01 0.22 0.64 0.29

* Values are least square means. Body weight was measured after an overnight fast. Body fat, LBM, BCM, TBW, and ICW were measured with bio-electrical impedance analysis.† P values for treatment effects were determined with a 23 2 factorial analysis of variance.‡ Pooled standard error of the mean.BCM, body cell mass; CR, creatine; CR3 HMB, interaction between creatine andb-hydroxy-b-methylbutyrate; CR/HMB, creatine plusb-hydroxy-b-methylbutyrate; HMB,b-hydroxy-b-methylbutyrate; ICW, intracellular water; LBM, lean body mass; TBW, total body water


Statistical Analysis

The data were evaluated by two-way factorial analysis of varianceby using the general linear model procedure of the StatisticalAnalysis System.21 In Tables III, IV, V, and VI, the data arepresented as the mean6 standard error of the mean for eachparameter, and the significance for the main effects of CR, HMB,and CR 3 HMB are given. When a significant CR3 HMBinteraction occurred, individual means were tested witht tests.Statistical significance was set atP , 0.05. A trend was set atP ,0.10.

RESULTS

Dietary Intake

The calculated intakes of protein, fat, and calories are presented inTable III. Overall protein intake was approximately 180% of therecommended daily allowance in all groups.22 Less protein tendedto be consumed by those supplemented with CR in week 2 (CRmain effectP , 0.08) and was significantly decreased in week 3

of the study (CR main effectP , 0.04). In addition, the caloricintake of the CR-supplemented subjects was significantly lowerthan that of subjects not supplemented with CR in week 2 (CRmain effectP 5 0.001) and tended to be lower in week 3 (CR maineffect P 5 0.07).

Body Composition

Body weight and composition changes are presented in Table IV.Body weight increased in all groups over the 3-wk period. CR andHMB supplements significantly increased body weight (treatmentmain effectsP 5 0.0001 and 0.007, respectively), with the com-bination of CR/HMB producing the greatest response. Increases inLBM were seen in all groups over the 3-wk period (Table IV). Theplacebo group had a gain of 0.85 kg (1.35%) compared with 1.77kg (2.8%) in the CR group, 1.24 kg (2.0%) in the HMB group, and2.39 kg (3.8%) in the CR/HMB group. Supplementing with CRsignificantly increased LBM (CR main effectP 5 0.0005), andHMB supplementation tended to increase LBM gain in 3 wk(HMB main effectP 5 0.08). The effects of CR and HMB tendedto be additive in that there was no interaction (CR3 HMB main

TABLE V.

MAXIMUM MUSCLE STRENGTH (1-RM) BEFORE AND AFTER 3 WK OF EXERCISE IN SUBJECTS SUPPLEMENTED WITH PLACEBO(n 5 10), CR (n 5 11), HMB (n 5 9), OR CR/HMB (n 5 10)

Treatment P†

Placebo CR HMB CR/HMB CR HMB CR3 HMB SEM‡

Bench press (kg)Screen 87.78 70.00 75.63 84.44 0.37 0.75 0.03 5.79Week 3 91.67 78.00 86.25 95.00 0.60 0.27 0.06 5.75Net 13.89 18.00 110.60 110.56 0.11 0.002 0.12 1.31

Power clean (kg)Screen 107.78 122.50 122.50 130.00 0.24 0.26 0.71 9.51Week 3 113.89 142.50 136.88 153.33 0.02 0.10 0.53 9.68Net 16.11 120.00 114.38 123.33 0.001 0.10 0.46 3.30

BTN (kg)Screen 50.83 44.50 51.19 48.89 0.28 0.54 0.62 3.99Week 3 54.17 51.50 61.25 58.89 0.56 0.09 0.97 4.20Net 13.33 17.00 110.06 110.00 0.20 0.003 0.21 1.47

Biceps curl (kg)Screen 46.67 41.50 44.06 45.00 0.46 0.84 0.33 3.07Week 3 48.89 47.25 49.38 51.33 0.98 0.43 0.55 2.99Net 12.22 15.75 15.31 16.33 0.008 0.04 0.14 0.84

Squat (kg)Screen 98.33 84.00 93.13 95.00 0.33 0.63 0.24 6.81Week 3 101.11 95.50 105.94 108.89 0.82 0.18 0.54 6.84Net 12.78 111.50 112.81 113.89 0.01 0.005 0.06 2.00

Triceps extension (kg)Screen 40.00 30.25 30.31 35.33 0.36 0.53 0.02 2.99Week 3 41.39 35.25 36.00 42.83 1.00 0.63 0.04 2.98Net 11.39 15.00 15.69 17.50 0.01 0.003 0.40 1.06

Accumulative (kg)Screen 431.39 392.75 416.81 438.67 0.71 0.52 0.26 26.59Week 3 451.11 450.00 475.69 520.28 0.56 0.12 0.52 27.31Net 119.72 157.25 158.87 171.61 0.001 0.001 0.10 7.27

* Values are least-square means.† P values for treatment effects were determined with a 23 2 factorial analysis of variance.‡ Pooled standard error of the mean.1-RM, one-repetition maximum; BTN, behind-the-neck press; CR, creatine; CR3 HMB, interaction between creatine andb-hydroxy-b-methylbutyrate;CR/HMB, creatine plusb-hydroxy-b-methylbutyrate; HMB,b-hydroxy-b-methylbutyrate


effect P 5 0.73) and the effect of the combination was differentfrom the sum of their seperate effects (Fig. 1a).

CR/HMB supplementation resulted in a synergistic increase inbody fat. There tended to be an interaction between CR and HMB(CR 3 HMB main effectP 5 0.06) as shown by the significantgain in body fat in the CR/HMB-supplemented group comparedwith the other groups (P , 0.05).

BCM, TBW, and ICW increased in all groups: 1.7%, 2.0%, and1.7%, respectively, in the placebo group compared with 3.9% (P ,0.01), 3.7% (P , 0.001), and 3.9% (P , 0.01) in the CR group,2.4%, 2.5%, and 2.4% in the HMB group, and 3.8%, 4.6%, and5.6% in the CR/HMB group. Increases in BCM, TBW, and ICWin the CR/HMB-supplemented group were additive (CR3 HMBmain effectsP 5 0.65, 0.91, and 0.64, respectively).

Muscle Strength

Maximum muscle strength (1-RM) before and after 3 wk ofexercise is shown in Table V. The accumulative 1-RM strengthincreases in the CR-, HMB- and CR/HMB-supplemented groups

were 37.5, 39.1, and 51.9 kg, respectively, above those of theplacebo group after 3 wk (CR and HMB main effectP 5 0.001).The strength increase in the CR/HMB group showed an additiveeffect over CR or HMB supplementation alone (Fig. 1b; CR3HMB effect P 5 0.10). At the beginning of the study, subjects inthe placebo group were stronger than those in the other groupsaccording to the bench-press and triceps-extension exercises.HMB supplementation resulted in significant (P , 0.05) increasesin strength in all the exercises measured except the power clean,which showed a trend for an increase in strength compared withplacebo supplementation. CR supplementation significantly (P ,0.05) increased strength gains for all exercises except the benchpress and behind-the-neck press compared with placebosupplementation.

Serum CPK, Creatinine, and Blood Urea Nitrogen

Changes in serum CPK, creatinine, and blood urea nitrogen areshown in Table VI. Serum CPK levels increased after 2 wk ofexercise in the placebo, CR-, and CR/HMB-supplemented groups

TABLE VI.

SERUM CPK, CRN, BUN, UCRN, AND UUN IN SUBJECTS SUPPLEMENTED WITH PLACEBO (n 5 10), CR (n 5 11), HMB (n 5 9), ORCR/HMB (n 5 10)

Treatment* P†

SEM‡Placebo CR HMB CR/HMB CR HMB CR3 HMB

CPK (U/L)Screening 207 430 372 230 0.37 0.54 0.001 51Week 2 1107 1186 421 833 0.26 0.01 0.39 190Week 3 453 459 282 396 0.35 0.10 0.43 68D2 949 756 38 539 0.40 0.01 0.08 193D3 223 29 290 162 0.50 0.21 0.0004 56

CRN (mg/dL)Screening 1.22 1.22 1.18 1.13 0.60 0.15 0.61 0.045Week 2 1.23 1.40 1.22 1.33 0.003 0.37 0.49 0.045Week 3 1.22 1.51 1.22 1.47 0.0001 0.70 0.71 0.057D2 0.01 0.18 0.04 0.20 0.002 0.60 0.87 0.049D3 0.00 0.29 0.04 0.36 0.0001 0.30 0.85 0.056

BUN (mg/dL)Screening 14.5 16.5 13.2 12.6 0.69 0.11 0.42 1.62Week 2 15.8 15.5 10.4 11.4 0.89 0.02 0.73 1.85Week 3 18.3 17.5 9.8 9.8 0.97 0.0001 0.80 1.72D2 1.30 20.90 22.78 21.20 0.81 0.23 0.29 1.76D3 3.33 20.40 23.44 23.33 0.39 0.02 0.34 1.98

UCRN (g/d)Screening 1.87 1.81 1.80 1.65 0.47 0.39 0.74 0.138Week 2 1.72 2.17 1.84 1.80 0.20 0.42 0.14 0.166Week 3 1.13 1.52 1.10 1.54 0.002 0.99 0.81 0.126D2 20.15 0.36 0.04 0.15 0.04 0.91 0.19 0.150D3 20.71 20.30 20.70 20.13 0.0009 0.50 0.57 0.133

UUN (g/d)Screening 11.0 8.9 11.5 8.6 0.02 0.96 0.67 1.03Week 2 9.0 9.7 10.4 5.4 0.04 0.10 0.005 0.95Week 3 11.5 9.7 6.7 6.4 0.29 0.0001 0.36 0.83D2 21.96 0.75 21.06 23.17 0.73 0.18 0.05 1.19D3 0.70 0.80 24.77 22.37 0.16 0.0001 0.25 0.98

* Values are least square means.† P values for treatment effects were determined with a 23 2 factorial analysis of variance.‡ Pooled standard error of the mean.BUN, blood urea nitrogen; CPK, creatine phosphokinase; CR, creatine; CR3 HMB, interaction between creatine andb-hydroxy-b-methylbutyrate;CR/HMB, creatine plusb-hydroxy-b-methylbutyrate; CRN, creatine;D2, change from screening to week 2;D3, change from screening to week 3;HMB, b-hydroxy-b-methylbutyrate; UCRN, urinary creatinine; UUN, urinary urea nitrogen


FIG. 2. Changes in plasma CPK levels for the placebo, HMB-, CR-, and CR/HMB-supplemented groups over 3 wk of progressive resistance training. Dataare least-square means.*P , 0.01 across groups. CPK, creatine, phosphokinase; CR, creatine; HMB,b-hydroxy-b-methylbutyrate.

FIG. 1. Effects of HMB and CR alone and in combination on LBM (a) and strength (b). CR and HMB when supplemented independently and in combinationhad significant effects on LBM and strength (P # 0.05). No significant CR3 HMB interaction was present for LBM or strength increase (CR3 HMB effect:P 5 0.73 and 0.10, respectively) indicating that CR and HMB were additive in relation to LBM and strength. CR, creatine; HMB,b-hydroxy-b-methylbutyrate; LMB, lean body mass.


but not in the HMB-supplemented group (Fig. 2). These changesresulted in a significant HMB effect (P 5 0.01) and a trend for aninteraction when CR also was supplemented (P 5 0.08). By week3, CPK levels returned to baseline values in the CR-supplementedgroup, remained elevated in the placebo and CR/HMB-supplemented groups, and decreased below screening values in theHMB-supplemented group, resulting in an interaction (CR3HMB effect P 5 0.004). These data suggest that CR antagonizedthe CPK-lowering effect of HMB. Supplementation with CR re-sulted in a significant increase in serum creatinine levels at weeks2 (CR effectP 5 0.002) and 3 (CR effectP 5 0.0001) comparedwith subjects not supplemented with CR. There were no effects ofHMB supplementation at 2 and 3 wk. Blood urea nitrogen de-creased by 26% in the HMB-supplemented groups at wk 3 (HMBmain effectP 5 0.02); CR alone had no significant effect on bloodurea nitrogen.

Urinary Creatinine and Urinary Urea Nitrogen

The levels of urinary creatinine and urea nitrogen for all subjectsare shown in Table VI and Fig. 3. Urinary creatinine was signif-icantly increased in the subjects supplemented with CR comparedwith subjects not supplemented with CR (CR main effectsP 50.04 and 0.0009 for weeks 2 and 3, respectively). Excretion ofurinary urea nitrogen was decreased synergistically by week 2 andsupported by a significant CR3 HMB interaction (P 5 0.05).However, by week 3, the synergistic effect was not significant.HMB supplementation continued to cause a significant decrease inexcretion of urinary urea nitrogen through week 3 (HMB maineffect P 5 0.0001).

DISCUSSION

The major finding of this research was that supplementation withCR and HMB was additive with regard to increasing LBM andstrength. These observations are consistent with, but do not prove,the hypothesis that CR and HMB act through distinct mechanisms.

HMB had an acute effect on LBM that was about half themagnitude of CR. The increase in LBM in the CR/HMB group washigher than that in the CR and HMB groups (2.39 kg versus 1.77kg and 1.24 kg, respectively). The increases due to CR and HMBsupplementation were additive because there was no significantinteraction (Fig. 1a).

The strength increase in the CR/HMB group also was shown tobe additive (CR3 HMB effect P 5 0.10), thus supporting theconcept that CR and HMB have different mechanisms of increas-ing strength (Fig. 1b). Greater strength according to the bench-press and triceps-extension exercises was observed in the placebogroup at screening and appeared to be a random-chance occurrencebecause the other strength measurements were not significantlyhigher in the placebo group than in the other groups.

The mechanism whereby HMB increases LBM and strength isproposed to be through a net increase in muscle-protein synthesisbrought about by a decrease in protein degradation. Although notdirectly related to muscle-protein turnover, previous studies haveshown that reduced protein degradation is accompanied by a lowerserum-CPK level, CPK being an indicator of muscle-cell mem-brane damage.15–17 In this study, there was a significantly lowerincrease in serum CPK in week 2 with HMB supplementation(HMB effect P 5 0.01) and a numerically lower week-3 serum-CPK concentration (HMB effectP 5 0.21) in comparison with the

FIG. 3. Urine urea nitrogen and plasma urea nitrogen changes for placebo, HMB-, CR-, and CR/HMB-supplemented groups from the initial screening toweek 3 of a progressive resistance-training regimen. Data are from the changes in the means. *P , 0.02 across groups. CR, creatine; HMB,b-hydroxy-b-methylbutyrate.


non–HMB-supplemented groups. In addition, the 42% decrease inurinary urea nitrogen and 26% decrease in serum urea nitrogenwith HMB supplementation (Fig. 3) supports the idea of increasednet-protein deposition.

CR supplementation has been proposed to increase LBM byincreasing osmotic pressure in muscle, which increases the watercontent of the muscle.1,4,10,11In this study, a significant increase inBCM, TBW, and ICW was shown in all groups supplemented withCR. There also were similarities in all groups between the per-centage of LBM gain and percentage of BCM, TBW, and ICWincreases. In vitro studies have shown that the rate of [3H]leucineincorporation into heavy-chain myosin increases in skeletal andcardiac muscles with exposure to CR but that total protein synthe-sis does not change.12–14The increase in muscle strength and LBMthat occurred with CR supplementation in this study may havebeen due to this specific increase in muscle-protein synthesis. Theurine-urea data (Table VI) suggest that CR supplementation didnot affect nitrogen retention. However, urine urea nitrogen alonecan be difficult to interpret because of relative nitrogen shifts whenseparating urea from ammonia, which can result in changes inurine urea without changes in total nitrogen output. However, CRmetabolism is not known to affect the shift between urea andammonia, so it is unlikely that CR caused a net increase of nitrogenand most unlikely that it affected net-protein synthesis.

Serum creatinine concentration increased by about 17% inweek 2 in the CR-supplemented groups and remained elevated forthe length of the study. Creatinine has been established as the soleend product of CR degradation because it is formed non-enzymatically in an irreversible chemical reaction.2,23 The raisedserum creatinine results from increased muscular CR. Urinarycreatinine increased in the CR-supplemented groups in week 2 asa result of increased serum creatinine.

CR alone had no effect on CPK levels and seemed to reversethe protective effect of HMB to about one-half of HMB alone (Fig.2). This loss of HMB’s ability to protect against the exercise-induced rise in CPK when combined with CR could be the resultof mechanisms different from those hypothesized for CR andHMB.

Dietary analysis of the different groups indicated that the CRgroups had lower caloric intakes of 13% to 20% in week 2 and 7%to 10% in week 3 in comparison with the placebo and HMBgroups. Although a lower caloric intake should result in a negativeenergy balance, fat mass was actually increased in the CR-supplemented groups compared with the placebo and HMB-supplemented groups. Protein intake was also 6% to 14% lower inthe CR groups than the placebo and HMB groups in week 3.However, total protein intake in all groups was approximately180% of the recommended daily allowance, suggesting that theslightly lower protein intake was still well above that required.

In conclusion, we found that HMB and CR increase LBM andstrength in an additive manner during a progressive resistance-training regimen. Although the present data did not determine theexact mechanisms of CR and HMB action, they are consistant withpreviously theoreticized mechanisms, i.e., that HMB increasesLBM by slowing muscle-protein breakdown, whereas CR in-creases LBM by increasing cellular water content.

ACKNOWLEDGMENTS

We thank S. M. Baier, J. C. Fuller Jr. and R. H. Minnion for theircollaboration in this investigation.

REFERENCES

1. Balsom PD, Soderlund K, Ekblom B. Creatine in humans with special referenceto creatine supplementation. Sports Med 1994;18:268

2. Bloch K, Schoenheimer R. The metabolic relation of creatine and creatininestudied with isotopic nitrogen. J Biol Chem 1939;131:111

3. Callmer E, Hagman U, Haraldsdottir J. Proposal for the standardization of24-hour recall and similar interview methods. Var Foda 1986;4(suppl):259

4. Clarkson PM, Rawson ES. Nutritional supplements to increase muscle mass. CritRev Food Sci Nutr 1999;39:317

5. Earnest CP, Snell PG, Rodriguez R, Almada AL, Mitchell TL. The effect ofcreatine monohydrate ingestion on anaerobic power indices, muscular strengthand body composition. Acta Physiol Scand 1995;153:207

6. Florenz AE. Body composition, body composition analyzer, version 2.1. 1998:17. Greenhaff PL, Bodin K, Soderlund K, Hultman E. Effect of oral creatine

supplementation on skeletal muscle phosphocreatine resynthesis. Am J Physiol1994;266:E725

8. Harris RC, Soderlund K, Hultman E. Elevation of creatine in resting and exer-cised muscle of normal subjects by creatine supplementation. Clin Sci (Colch)1992;83:367

9. Hultman E, Soderlund K, Timmons JA, Cederblad G, Greenhaff PL. Musclecreatine loading in men. J Appl Physiol 1996;81:232

10. Kreider RB. Dietary supplements and the promotion of muscle growth withresistance exercise. Sports Med 1999;27:97

11. Stone MH, Sanborn K, Smith LL, et al. Effects of in-season (5 weeks) creatineand pyruvate supplementation on anaerobic performance and body compositionin American football players. Int J Sport Nutr 1999;9:146

12. Ingwall JS, Morales MF, Stockdale FE. Creatine and the control of myosinsynthesis in differentiating skeletal muscle. Proc Natl Acad Sci USA 1972;69:2250

13. Ingwall JS, Wildenthal K. Creatine stimulation of myofibrillarprotein synthesis.J Cell Biol 1974;63:151A

14. Ingwall JS, Wildenthal K. Role of creatine in the regulation of cardiac proteinsynthesis. J Cell Biol 1976;68:159

15. Nissen S, Sharp R, Ray M, et al. The effect of the leucine metaboliteb-hydroxyb-methylbutyrate on muscle metabolism during resistance-exercise training.J Appl Physiol 1996;81:2095

16. Nissen SL, Abumrad NN. Nutritional role of the leucine metaboliteb-hydroxy-b-methylbutyrate (HMB). J Nutr Biochem 1997;8:300

17. Panton LB, Rathmacher JA, Baier S, Nissen S. Nutritional supplementation of theleucine metabolite B-hydroxy B-methylbutyrate (HMB) during resistance train-ing. Nutrition 2000;16:734

18. Baumgartner RN, Chumlea WC, Roche AF. Bioelectric impedance for bodycomposition. Exerc Sport Sci Rev 1990;18:193

19. Shizgal HM. Validation of the measurement of body composition from wholebody bioelectric impedance. Infusionstherapie 1990;17(suppl):67

20. Bioelectrical impedance analysis in body composition measurement: NationalInstitutes of Health Technology Assessment Conference Statement. Am J ClinNutr 1996;64(suppl):524S–532S.

21. SAS Institute.SAS user’s guide on statistics. Cary, NC: SAS Institute, 198522. Subcommittee on the Tenth Edition of the RDAs.Recommended dietary allow-

ances. Washington, DC: National Academy Press, 198923. Hunter A. The physiology of creatine and creatinine. Physiol Rev 1922;2:586


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Nutr (2001) Creatine, HMB and Hypertrophy