ADVANCING age leads to a decrease in skeletal muscle

The aim of the present study was to investigate the effect of protein and carbohydrate ingestion immediately after an acute resistance exercise session on leg muscle protein balance and kinetics. We investigated the effect of nutrient administration after a session of resistance exercise on muscle protein kinetics in six healthy, early postmenopausal women. Each participant was tested twice, at two different days, separated by at least a 4-week wash-out period.

1 Journal of Gerontology: MEDICAL SCIENCES 2005, Vol. 60A, No. 9, Copyright 2005 by The Gerontological Society of America Postexercise Nutrient Intake Enhances Leg Protein Balance in Early Postmenopausal Women Lars Holm, 1 Birgitte Esmarck, 2 Charlotte Suetta, 1 Keitaro Matsumoto, 3 Tatsuya Doi, 3 Masao Mizuno, 4 Benjamin F. Miller, 1 and Michael Kjaer 1 1 Institute of Sports Medicine, Copenhagen, Bispebjerg Hospital, Denmark. 2 Copenhagen Muscle Research Centre, Rigshospitalet, Denmark. 3 Saga Nutraceuticals Research Institute, Otsuka Pharmaceutical, Saga, Japan. 4 Research Unit 247, Ribe County Hospital Esbjerg, Esbjerg, Denmark. Background. We investigated the effect of nutrient administration after a session of resistance exercise on muscle protein kinetics in six healthy, early postmenopausal women, in a crossover design of random and double-blinded administration of protein and carbohydrate (PC) or placebo (NON). Methods. Fasted participants received a primed-constant infusion of L-[ring- 2 H 5 ]-phenylalanine. After 90 minutes of rest, the participants performed leg-resistance exercises followed by the oral supplementation. During the following 4 hours, net protein balance (NB) and rate of disappearance and appearance of phenylalanine were calculated from arterial venous blood samples and blood flow measurements. Results. NB was elevated ( p,.001) in the PC group compared to the NON group, and NB was not different from zero in the PC group, whereas it was negative in the NON group. Net balance results were supported by kinetic data from a reduced number of participants, showing that rate of disappearance was responsible for the initial (,1 hour) effect of PC, whereas a reduced rate of appearance enhanced the NB from 1.5 to 3 hours after training in the PC group. Conclusion. In early postmenopausal women, nutrient ingestion following resistance exercise improved anabolism by enhancing NB in skeletal muscle. ADVANCING age leads to a decrease in skeletal muscle mass (1), which may interfere with life quality and longevity (2). In general, women have less muscle mass than men, hence, an accelerated loss of muscle may become restrictive to their everyday function at an earlier age than men (3). Thus, middle-aged women are a high-priority target group for a preventative intervention toward functionrestricting sarcopenia. Middle-aged women also enter menopause, which is characterized by severe reduction of female sex hormones. Even though the exact role of the female sex hormones on muscle and lean body mass remains uncertain (4,5), many physiological changes are known to happen during these years, which might make menopausal women incomparable with women in other age groups. It is well established that muscle activity, at least in the form of heavy resistance exercise, improves muscle protein accretion by elevating protein-synthetic processes more than protein degradation (6), and that this ability is retained throughout age (7,8). Similarly, it is clear that protein retention at rest is enhanced in the postprandial situation compared with the fasted state (9,10). It is also well described that young individuals derive an acute anabolic advantage when combining resistance exercise and nutritional intake (11 13). However, the muscle protein responsiveness in middle-aged, postmenopausal women to this combination has not been investigated. Some studies (9,14,15) indicate that the nutritional responsiveness may be lower with increasing age. Further studies are needed to distinguish exercise and nutrition interactions in an effort to preserve muscle strength, and thus, functional ability in this susceptible population. The aim of the present study was to investigate the effect of protein and carbohydrate ingestion immediately after an acute resistance exercise session on leg muscle protein balance and kinetics in early postmenopausal women who are not using hormone substitution. METHODS Participant Selection Women were recruited from a newspaper advertisement. During the initial interview, women with known myoskeletal disorders, frequent use of medication, and history of resistance exercise training in the year prior to the study were not invited for further investigation. After an initial interview, 10 women were invited to a physical examination, and a 12-hour fasting blood sample, which was screened for substances (such as immunocytes, electrolytes, creatinine, enzymes, glucose, and lipids) that could indicate presence of different metabolic disorders. Two of the women were rejected, and the remaining eight completed a VO 2 max test on a stationary bike (Bikerace HC600; Technogym, Gambettola, Italy). During the VO 2 max test, blood pressure (Baumanometer, 300 model; W. A. Baum Co. Inc., New York, NY), electrocardiogram (Nihon Kohden Electrocardiograph ECG-9329K; Tokyo, Japan), and oxygen uptake (Innovision A/S model AMIS2001; Odense, Denmark) were measured continuously until voluntary fatigue. Two participants were disqualified for irregular electrocardiograms, so a total of six healthy early postmenopausal women were included in the study. The study was approved by the local 1212

2 POSTEXERCISE NUTRIENTS IN AGING WOMEN 1213 variances in energy expenditure. Adequate protein intake was set to 0.8 g/kg/day [lowest daily recommendation for adults with a limited level of physical activity (16)]. Figure 1. Arterial plasma concentration of phenylalanine (Phe) (A) and essential amino acids (EAA) (B). Values are mean; bars are standard error of the mean (SEM). Squares represent the protein and carbohydrate (PC) group; triangles represent the placebo (NON) group. ### denotes interaction ( p,.0001) in a two-way analysis of variance with repeated measures analysis. Ethical Committee of Copenhagen (KF) /01 and, in accordance with the Declaration of Helsinki, the study protocol, purpose, and possible risks were explained to each participant before their written consent was obtained. Dietary Control Participants completed a weighed-food record on 4 nonconsecutive days prior to the first trial. The food recordings were analyzed on Ankerhus software (Winfood, version 2.0; Ankerhus, Denmark) for daily energy and protein intake. The recorded amount of daily energy intake was compared to an estimation of adequate energy intake, which was calculated by multiplying an estimated value of the basal metabolic rate from the Harris-Benedict equation with an activity factor set to 1.6 (16). Adequate energy intake was set to.75% of calculated energy intake taking both the accuracy of the Harris-Benedict equation (17) and activity factor into consideration as well as individual Experimental Design and Protocol Each participant was tested twice, at two different days, separated by at least a 4-week wash-out period, using a double-blinded randomized crossover design with supplement administration of either 10 grams of protein (soy and milk protein), 31 grams of carbohydrate (dextrose), and 1 gram of fat, a total of 725 kj (PC), or an equal-tasting placebo product containing 6 grams of carbohydrate providing 100 kj (NON) (Otsuka Pharmaceuticals Co, Ltd., Saga, Japan) (Figure 1). At least 1 week prior to the first trial each participant determined their 10 repetition maximum (RM) for each exercise and was familiarized to the protocol. The exercise protocol consisted of three exercises: leg-press exercise in supine position with feet high, resulting in a squatlike exercise (3 sets times 10 repetitions at 10 RM); leg-press exercise with a low foot position, isolating quadriceps as the prime-mover (4 sets times 10 repetitions at 10 RM); and knee extension (4 sets times 10 repetitions at 10 RM). Two days prior to each trial the participants were instructed to avoid strenuous physical activities and caffeine, and to eat their normal meals. Participants fasted from 10 PM the previous night, with water allowed ad libitum during both fasting and the trial. The participants arrived in the laboratory at 7:30 AM by car and then rested in a bed. A catheter was inserted in the antecubital vein, and a basal blood sample was drawn. At 8:00 AM ( 120 minutes), a primed (3 lmol/kg), constant (0.05 lmol/kg/min) infusion of L-[ring- 2 H 5 ]phenylalanine (98% enriched; Cambridge Isotope Laboratories, Andover, MA) was initiated with a target tracer-to-tracee ratio of 5% 10% in arterial blood. Tracers were dissolved in sterile 0.9% saline solutions and filtered through a 0.2-lm sterile disposable filter before infusion. At 9:30 AM ( 30 minutes), the resistance exercise session was started. After 5 minutes of warming up on a cycle ergometer (cadence.60 rpm), the exercise protocol was conducted. All sets were conducted as fast as possible, with an interval of 2 minutes between each set with participants remaining passive on the training equipment. Immediately after the completion of training, the participants consumed the supplementation within 1 minute. Supplement ingestion was designated as time zero (0 minutes). A catheter was then inserted following local anesthetic in the right femoral artery using the Seldinger technique (18), with another catheter placed retrogradely in the femoral vein of the same leg. At 30, 60, 90, 120, 180, and 240 minutes, arterial and venous blood samples were drawn from the femoral catheters. Simultaneously, blood flow was determined in the contralateral femoral artery just above the bifurcation of the femoral artery by the ultrasound Doppler (Siemens, Ballerup, Denmark) technique (19). Analytical Procedures The arterial venous blood samples used for analysis of amino acid concentration, amino acid enrichment, and hormones were collected into 15% EDTA tubes (Vacutainer Systems, Plymouth, U.K.), spun at 5000 rpm for 15 minutes

3 1214 HOLM ET AL. at 48C, and immediately stored at 808C for later analysis. Glucose and hematocrit samples were collected as whole blood in lithium heparin and analyzed immediately on an ABL-700 series apparatus (Radiometer Medical A/S, Copenhagen, Denmark). Insulin plasma concentration was measured by an enzyme-linked immunosorbent assay (ELISA) kit (DAKO, Glostrup, Denmark). Estradiol concentrations were analyzed from basal blood samples after 8 hours of fasting using a competitive immunoassay (Immulite 2000; Diagnostic Products Corporation, Los Angeles, CA). Amino acid concentrations were determined from plasma samples (200 ll), which were deproteinized with 200 ll of 3% sulfosalicylic acid. The supernatant was assayed with an amino acid analyzer (L-8500; Hitachi, Tokyo, Japan) with S-(2-aminoethyl)-L-cysteine used as an internal standard. Plasma phenylalanine enrichment was determined from plasma samples as phenyl isothiocyanate (PICT) derivates (Fluka Chemie GmbH, Munich, Germany) by liquid chromatography mass spectrometry (Finnigan AQA, Manchester, U.K.) performed essentially as described elsewhere (20,21). After centrifugation (10,000 rpm for 4 minutes) of the plasma, 100 ll was mixed with 100 ll of internal standard (Norleucine, 98% enriched; Cambridge Isotope Laboratories, Andover, MA) in centrifugal filter devices (Ultrafree-MC; Millipore Corporation, Billerica, MA) and centrifuged for 45 minutes at 15,000 rpm. Coupling buffer (methanol/water/triethylamine, 2:2:1) and PICT derivatization solution (triethylamine/water/pict/methanol, 1:1:1:7) were applied separated by N 2 -drying. Finally, 100 ll of ammonium acetate buffer was applied. Calculations Protein kinetics. Net leg balance of phenylalanine was derived from an equation based on the Fick Principle: Net balance ¼ð½pheŠ a ½pheŠ v ÞBF ½1Š; where [phe] a and [phe] v are blood concentration of phenylalanine in arterial and venous blood, respectively, and BF is the blood flow supplying the limb. A positive value denotes net thigh uptake, and a negative value denotes net thigh release of the specific substrate, here phenylalanine, which is neither synthesized nor metabolized in the leg. It is assumed that muscle protein turnover primarily accounts for the leg metabolism of phenylalanine. Because the real precursor pool, aminoacylated trna, is not determined by this model, we have chosen an approach that does not attempt to estimate real kinetic values (22), but that asserts that the irreversible loss of tracee (Rate of disappearance, Rd) into the muscle-bed is estimated from the fractional extraction (FE) of its tracer from the blood: FE ¼ ½ð½pheŠ a E a Þ ð½pheš v E v ÞŠ ð½pheš a E a Þ 1 ½2Š; where E a and E v are the phenylalanine enrichment in arterial and venous blood. Thus: Rd ¼ FE E a BF ½3Š Using this approach, we find that the Rd value refers to the net amount of tracee and tracer disappearing from the arterial side into muscle tissue. This is only part of the real precursor, because recycling of tracee, directly as a product from breakdown to a precursor for synthesis, may take place intracellularly. Therefore, Rd is an underestimation of the real synthetic rate (22). Rate of appearance (Ra) of tracee into the blood now can be calculated by subtracting the NB from Rd: Ra ¼ Rd NB ½4Š Similarly, the Ra value represents the net amount of tracee that makes it into the blood (22). This is less than the total rate of production, due to recycling as described above. Hence, Rd is an underestimation of protein breakdown as well (22). Plasma phenylalanine concentrations are corrected to whole blood values by the hematocrit for calculation of NB, Rd, and Ra: Blood concentration ¼½pheŠ plasma ð100 hct%þ100 1 ½5Š; where [phe] plasma is the phenylalanine concentration in plasma, and hct% is the hematocrit as a percentage. Statistical analysis. Data are expressed as means 6 standard error of the mean. The effect of supplementation over time was evaluated by a two-way analysis of variance with repeated measures. A Bonferroni post hoc test was used to determine pairwise differences at individual time points when significant group interaction appeared. A t test was used to compare values to zero (dependent). Statistical significance was set at p,.05. Analyses were completed with Prism 4.0 (GraphPad Software, San Diego, CA). RESULTS Participant Characteristics The physical fitness (VO 2max ) averaged ml/kg/ min and body mass index kg/m 2, which are fairly normal for individuals at this age. One participant exercised regularly on either cycle ergometer or step-machine, whereas two others used a bike as daily transportation. The three remaining participants refrained from exercise except activity necessary during everyday life. Mean age of the women was years, and they averaged years since their last menstrual cycle. All participants had a plasma estradiol concentration below 0.10 nmol/l, indicating a ceased ovarian production of estradiol. Average daily energy intake was kj, which corresponded to the estimated daily need of kj. The average recorded daily protein intake was g, corresponding to values within the range g protein/kg body mass, which presumably is a sufficient amount of protein for this group of individuals to remain weight stable. Amino Acid Concentration, Enrichment, and Blood Flow Between trials there was an interaction effect ( p,.0001) for arterial phenylalanine concentration (Figure 1A) and

4 POSTEXERCISE NUTRIENTS IN AGING WOMEN 1215 Figure 2. Phenylalanine tracer enrichment in arterial plasma, n ¼ 4. Values are mean; bars are standard error of the mean (SEM). Squares represent the protein and carbohydrate (PC) group; triangles represent the placebo (NON) group. ### denotes time effect ( p,.001) in a two-way analysis of variance with repeated measures analysis. No difference appeared between trials. essential amino acid concentration (Figure 1B). However, no difference in arterial phenylalanine enrichment was apparent between trials, but there was a time effect ( p,.001) (Figure 2). Similarly, blood flow changed ( p,.05) over time from rest till 240 minutes with no difference between trials (Figure 3). Protein Kinetics Phenylalanine net balance values changed over time ( p,.001) and were elevated in the PC group compared to the NON group ( p,.001) (Figure 4A). Net balance values in the PC group were not different from zero ( p.05) in contrast to values in the NON group, which were less than zero ( p,.001). Figure 3. Blood flow in femoral arterial vessel. Values are mean; bars are standard error of the mean (SEM). Squares represent the protein and carbohydrate (PC) group; triangles represent the placebo (NON) group. # denotes time effect ( p,.05) in a two-way analysis of variance with repeated measures analysis. No difference appeared between trials. Figure 4. Net Balance of phenylalanine (A), phenylalanine rate of disappearance (Rd) an estimate of protein synthesis (B), and phenylalanine rate of appearance (Ra) an estimate of protein breakdown (C). Values are mean; bars are standard error of the mean (SEM). Squares represent the protein and carbohydrate (PC) group; triangles represent the placebo (NON) group. ### denotes time effect ( p,.001) and *** denotes supplementation effect ( p,.001) in a two-way analysis of variance with repeated measures analysis.

5 1216 HOLM ET AL. Because isotopic steady state is required for calculations of amino-acid kinetics, two of the participants were eliminated from this calculation due to wave-like fluctuations in arterial phenylalanine enrichment over time. Hence, kinetic data are calculated for four participants, who had linear, slightly elevating enrichment curves, indicating isotopic steady state at each individual measure point. On n ¼ 4, no statistical differences appeared for Rd and Ra (Figure 4, B and C). However, p values for interaction were.12 and.20 for Rd and Ra, respectively. Glucose and Insulin There was an interaction ( p,.001) between trials for arterial glucose concentration (data not shown) with most pronounced elevations at 60 and 90 minutes. Mean venous insulin concentration rose threefold in the PC group from fasting levels at rest to peak values at 60 minutes (interaction p,.0001) (Figure 5). DISCUSSION The present study demonstrated that ingestion of protein and carbohydrate immediately after a resistance exercise session elevated the net protein balance across the trained leg in middle-aged, early postmenopausal women when compared to placebo intake after resistance exercise. However, the response was attenuated when compared to previously published data on young individuals. Attenuated Response to Nutrients Following Exercise in Elderly Persons Until now no studies have used stable isotopic tracers and net balance to examine the acute response to the combination of exercise and nutrition in the elderly population. A previous study on elderly persons demonstrated greater muscle hypertrophy when supplementation is taken immediately following exercise compared to 2 hours after (23), indicating that timing of nutrient intake is important in older individuals. This finding is similar to those in studies of young persons that find that resistance exercise increases muscle protein turnover but that nutrient intake is necessary to take advantage of the full anabolic processes following resistance exercise (11 13), as net balance remains negative when fasting is sustained (6,11 13). Our results show that, in early postmenopausal women, resistance exercise does not increase net protein balance to positive values when fasting conditions are maintained (Figure 4A). However, with ingestion of 10 grams of protein and 31 grams of carbohydrate, protein balance is elevated and equals zero for up to 4 hours after exercise. Previous data from young persons indicate that ingestion of a comparable nutrient composition (6 grams of essential amino acids and 35 grams of glucose) after resistance exercise enhances net protein balance to positive values (12,24). Therefore, our results indicate that early postmenopausal women demonstrated an impaired, although elevated, responsiveness to nutrient intake. Although young participants were not compared to old participants in this investigation, the impaired responsiveness to mixed nutritional intake may help explain reduced lean body mass with increasing age. Figure 5. Venous insulin concentrations. Values are mean, bars are standard error of the mean (SEM). Squares represent the protein and carbohydrate (PC) group; triangles represent the placebo (NON) group. ### denotes interaction ( p,.0001) in a two-way analysis of variance with repeated measures analysis. We believe that previously published results in young and old persons lend support to the idea of an impaired nutritional response. First, it has been demonstrated that aging muscle retains acute responsiveness (i.e., increased turnover) to exercise (7,25) and retains ability to hypertrophy during long-term training (23,26,27), and that mixed muscle and myofibrillar protein synthesis rates after exercise are comparable to those of young individuals (7,25). Second, some degree of insulin resistance, which is frequently seen among elderly persons (28), may result in diminished nutrient responsiveness in old persons as compared to younger persons. The role of insulin must be considered because mixed meals (as in our study), as opposed to proteinonly meals, are more representative of daily food intake. Finally, although the overall time-dependent response to amino-acid ingestion may not vary between older and younger individuals, the anabolic response in the first hour following ingestion is attenuated in the older individuals (9). Such attenuation in the first hour following ingestion has been attributed to a greater first-pass extraction in the gut of older persons (9,29), which may be an effect of insulin resistance (30). The rapid increase in plasma and intracellular amino acid concentration could be crucial in the light of the proposed critical period of nutrient ingestion following exercise (23). Therefore, it appears that responsiveness to exercise (acute and chronic) is maintained, but attenuated sensitivity to insulin and an attenuated initial increase in plasma amino acids may account for the attenuated net balance response. However, it is important to note that despite the attenuated response (compared to the positive net balance in young participants) the early postmenopausal women still benefited from the addition of nutrient ingestion following exercise. Methodological Considerations We chose to use the two-pool model for calculation of substrate kinetics. Fundamentally, isotopic as well as physiological steady state is a prerequisite for the use of

6 POSTEXERCISE NUTRIENTS IN AGING WOMEN 1217 this model (22). Net protein balance was calculated from arterial venous blood samples and blood flow measurements, using the Fick Principle. To apply the Fick Principle, steady state must be present during the time intervals in which measurements are collected. If the time intervals are small (minutes/few hours), it is accepted (12,24,31) and reasonable to claim that steady state is present within each measurement (32) even though when larger (several hours) intervals are viewed, nonsteady state might appear. Therefore, by (a) determining blood flow as a mean of the pulsatory flow over repetitive cardiac cycles within a time frame no longer than 10 minutes, (b) collecting arterial blood prior to venous, and (c) calculating net balance as single values at individual time points and not as a mean of longer periods (hours), we believe that the prerequisite for using the Fick Principle for determining phenylalanine net balance across the leg was met. However, two participants were excluded from the kinetic calculations because their arterial tracer enrichment did not maintain a steady level throughout the 4-hour period. Amino Acid Kinetics and Net Balance As described above, it is known that the two-pool model underestimates the real values for protein synthesis and breakdown (22). However, in theory both variables are underestimated by the same value, i.e., the rate of intracellular recycling of tracee. Hence, the mutual relations between variables calculated with this approach should correspond to the real kinetic values. Although it must be considered that the phenylalanine kinetics are from an n of 4, the results are discussed in support of our net balance measurements. Ingestion of protein and carbohydrate tended ( p ¼.12) to elevate Rd (protein synthesis) in the exercised limb during the period in which amino acid availability was increased. This availability-dependent effect of amino acids, and especially essential amino acids on protein synthesis, is previously reported in young persons (12,24,33). In a recent study on resting elderly persons (9) a similar time course for arterial phenylalanine concentration and net balance was reported following oral ingestion of 15 grams of amino acids. As discussed above, compared to the response observed in younger individuals, the aged individuals seem to have a slower and more prolonged response (9). Because in the present study net balance is not different from zero in the initial period following ingestion, early postmenopausal women may respond more similarly to elderly participants than to younger participants. In the present study, in which carbohydrate and protein was ingested, it was expected that Ra (protein breakdown) would be diminished (15). After exercise and PC, phenylalanine Ra peaked at 30 minutes and was equal to NON by 60 minutes. However, the Rd increased sufficiently to maintain net balance not different to zero, whereas in the NON group, Ra exceeded Rd resulting in negative net balance throughout the period. In the PC group, insulin concentration peaked at 60 minutes after ingestion. Because insulin is known to have a potent, postponed anticatabolic effect (34), it is interesting that the decreased Ra at 60 minutes preceded the insulin peak at 60 minutes, and that no significant change in Ra in the period following the insulin peak appeared. As discussed above, a decreased insulin response (either centrally or peripherally) could account for the lack of significant change in Ra and the attenuated net balance response in the early postmenopausal women compared to data from younger participants. However, as with net balance, it is important to realize that there was indeed a positive effect of PC ingestion compared to NON following exercise. Conclusion The present study demonstrated that early postmenopausal women did benefit acutely from ingestion of protein and carbohydrate immediately after a resistance exercise session to increase skeletal muscle protein accretion. This finding provides the basis for a long-term effect of such nutrient intake in combination with exercise training in postmenopausal women in counteracting muscle loss with aging. ACKNOWLEDGMENTS We thank the participants for their attendance in this study, and we are grateful for the technical assistance of Annie Høj, Birgitte Lillethorup, Ann- Marie Sederstrøm, and Ann-Christina Henriksen during the studies and with analysis of samples. The study was supported by Otsuka Pharmaceuticals Co. Ltd., Saga, Japan. Address correspondence to Lars Holm, Institute of Sports Medicine, Copenhagen, Bld. 8 1st Bispebjerg Bakke 23, 2400 Copenhagen NV, Denmark. lh17@bbh.hosp.dk REFERENCES 1. Klitgaard H, Mantoni M, Schiaffino S, et al. Function, morphology and protein expression of ageing skeletal muscle: a cross-sectional study of elderly men with different training backgrounds. Acta Physiol Scand. 1990;140: Schechtman KB, Ory MG. The effects of exercise on the quality of life of frail older adults: a preplanned meta-analysis of the FICSIT trials. Ann Behav Med. 2001;23: Kamel HK, Maas D, Duthie EH Jr. Role of hormones in the pathogenesis and management of sarcopenia. 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