Amino acid absorption and subsequent muscle …...Amino acid absorption and subsequent muscle protein accretion following graded intakes of whey protein in elderly men Bart Pennings,1,2

Amino acid absorption and subsequent muscle protein accretion followinggraded intakes of whey protein in elderly men

Bart Pennings,1,2 Bart Groen,1,2 Anneke de Lange,2 Annemie P. Gijsen,3 Antoine H. Zorenc,2

Joan M. G. Senden,1,2 and Luc J. C. van Loon1,2

1Top Institute Food & Nutrition, Wageningen; and Departments of 2Human Movement Sciences and 3Human Biology,NUTRIM School for Nutrition, Toxicology, and Metabolism, Maastricht University, Maastricht, The Netherlands

Submitted 11 October 2011; accepted in final form 5 February 2012

Pennings B, Groen B, de Lange A, Gijsen AP, Zorenc AH, SendenJMG, van Loon LJC. Amino acid absorption and subsequent muscleprotein accretion following graded intakes of whey protein in elderlymen. Am J Physiol Endocrinol Metab 302: E992–E999, 2012. Firstpublished February 14, 2012; doi:10.1152/ajpendo.00517.2011.—Wheyprotein ingestion has been shown to effectively stimulate postprandialmuscle protein accretion in older adults. However, the impact of theamount of whey protein ingested on protein digestion and absorptionkinetics, whole body protein balance, and postprandial muscle proteinaccretion remains to be established. We aimed to fill this gap byincluding 33 healthy, older men (73 � 2 yr) who were randomlyassigned to ingest 10, 20, or 35 g of intrinsically L-[1-13C]phenylala-nine-labeled whey protein (n � 11/treatment). Ingestion of labeledwhey protein was combined with continuous intravenous L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosine infusion to assess themetabolic fate of whey protein-derived amino acids. Dietary proteindigestion and absorption rapidly increased following ingestion of 10,20, and 35 g whey protein, with the lowest and highest (peak) valuesobserved following 10 and 35 g, respectively (P � 0.05). Whole bodynet protein balance was positive in all groups (19 � 1, 37 � 2, and58 � 2 �mol/kg), with the lowest and highest values observedfollowing ingestion of 10 and 35 g, respectively (P � 0.05). Post-prandial muscle protein accretion, assessed by L-[1-13C]phenylalanineincorporation in muscle protein, was higher following ingestion of 35g when compared with 10 (P � 0.01) or 20 (P � 0.05) g. We concludethat ingestion of 35 g whey protein results in greater amino acidabsorption and subsequent stimulation of de novo muscle proteinsynthesis compared with the ingestion of 10 or 20 g whey protein inhealthy, older men.

nutrition; aging; sarcopenia; fractional synthetic rates; stable isotopes

AGING IS ACCOMPANIED BY a progressive decline in skeletalmuscle mass, termed sarcopenia (15). Data have been obtainedto suggest that the skeletal muscle protein synthetic response tofood intake is impaired in older adults (7, 12). This proposedanabolic resistance is now regarded a key factor in the etiologyof sarcopenia. Consequently, we and other laboratories havebeen trying to define effective interventional strategies tocompensate for this anabolic resistance by improving postpran-dial muscle protein accretion in older adults (14, 20, 22, 23).Recently, we assessed the impact of protein digestion andabsorption kinetics as well as amino acid composition onpostprandial muscle protein synthesis rates in older men. Theresults showed that whey protein was more effective thaneither casein or casein hydrolysate protein in stimulating post-prandial muscle protein accretion (19).

Besides the quality of dietary protein, i.e., its amino acidcomposition and digestion and absorption kinetics, also theamount of dietary protein ingested likely modulates postpran-dial muscle protein synthesis rates (14, 19). So far, no studyhas assessed the impact of ingesting different amounts of wheyprotein on protein digestion and absorption kinetics, wholebody protein balance, and postprandial muscle protein synthe-sis rates in older adults. Because the metabolic fate of aminoacids ingested as dietary protein cannot be assessed by oral orintravenous administration of labeled free amino acids (6, 8),we specifically produced intrinsically labeled whey protein byinfusing cows with large quantities of L-[1-13C]phenylalanine,collecting milk, and purifying the whey protein fraction (21).The use of intrinsically labeled whey protein allowed us toassess the impact of ingesting different amounts of wheyprotein on in vivo protein digestion and absorption kinetics andsubsequent muscle protein accretion without the need forextensive assumptions and extrapolations.

In the present study, 33 elderly men ingested a single bolusof 10, 20, or 35 g intrinsically L-[1-13C]phenylalanine-labeledwhey protein. Ingestion of labeled whey protein was combinedwith continuous intravenous L-[ring-2H5]phenylalanine andL-[ring-2H2]tyrosine infusion, during which blood and muscletissue samples were collected. This study is the first to describethe impact of the amount of whey protein ingested on subse-quent protein digestion and absorption kinetics, splanchnicsequestration, whole body protein metabolism, and postpran-dial muscle protein accretion in vivo in older males.

MATERIALS AND METHODS

Participants. Thirty-three healthy, older men (73 � 2 yr) partici-pated in this study. Subjects were randomly assigned to ingest a singlebolus of 10, 20, or 35 g intrinsically L-[1-13C]phenylalanine-labeledwhey protein (n � 11/treatment). Subjects’ characteristics are pre-sented in Table 1. None of the subjects had a history of participatingin any regular exercise program. All subjects were informed on thenature and possible risk of the experimental procedures before theirwritten informed consent was obtained. This study was approved bythe Medical Ethics Committee of the Academic Hospital Maastricht.

Pretesting. Before selection in the study, an oral glucose tolerancetest (OGTT) was performed to assess glucose tolerance and screen fortype 2 diabetes prevalence according to World Health Organizationcriteria (2). Before the OGTT, body weight and height were assessed,and body composition was determined by DXA (Discovery A; Ho-logic, Bedford, MA).

Diet and activity before testing. All subjects consumed a standard-ized meal (32 � 2 kJ/kg body wt, providing 55 energy/100% energycarbohydrate, 15 energy/100% energy protein, and 30 energy/100%energy fat) the evening before the experiment. All volunteers wereinstructed to refrain from any sort of exhaustive physical activity and

Address for reprint requests and other correspondence: L. J. C. van Loon,Dept. of Human Movement Sciences, Maastricht Univ., P.O. Box 616, 6200MD, Maastricht, The Netherlands (e-mail: [email protected]).

Am J Physiol Endocrinol Metab 302: E992–E999, 2012.First published February 14, 2012; doi:10.1152/ajpendo.00517.2011.

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to keep their diet as constant as possible 3 days before the experi-ments.

Experimental protocol. At 8:00 A.M., following an overnight fast,subjects arrived at the laboratory by car or public transportation. ATeflon catheter was inserted in an antecubital vein for stable isotopeinfusion. A second Teflon catheter was inserted in a heated dorsalhand vein of the contralateral arm and placed in a hot box (60°C) forarterialized blood sampling (1). Following basal blood collection[time (t) � �240 min], the plasma phenylalanine and tyrosine poolswere primed with a single intravenous dose of L-[ring-2H5]pheny-lalanine (2 �mol/kg) and L-[ring-2H2]tyrosine (0.775 �mol/kg), afterwhich continuous L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosineinfusion was started (0.050 � 0.001 and 0.019 � 0.001�mol·kg�1·min�1, respectively). After resting in a supine position for120 min, a second arterialized blood sample was drawn, and the firstmuscle biopsy was collected from the vastus lateralis muscle (t ��120 min), marking the start of a fasted, baseline period. During thisperiod, additional blood samples were drawn every 30 min, and asecond muscle biopsy was taken at t � 0 min. Directly following thesecond biopsy, subjects ingested a single bolus of 10, 20, or 35 gintrinsically L-[1-13C]phenylalanine-labeled whey protein dissolved in400 ml water, which was uniformly flavored by adding 5 ml vanillaflavor (Givaudan, Naarden, The Netherlands) per liter beverage.Arterialized blood samples were collected at t � 15, 30, 45, 60, 90,120, 180, and 240 min with a third muscle biopsy taken from thecontralateral limb at t � 240 min. Blood samples were collected inEDTA-containing tubes and centrifuged at 1,000 g for 5 min at 4°C.Aliquots of plasma were frozen in liquid nitrogen and stored at�80°C. Muscle biopsies were obtained from the middle region of thevastus lateralis. Biopsies from the same incision were taken in a distaland proximal direction, respectively. Muscle samples were dissectedcarefully and freed from any visible nonmuscle material. The musclesamples were immediately frozen in liquid nitrogen and stored at�80°C until further analysis.

Preparation of intrinsically labeled whey protein. IntrinsicallyL-[1-13C]phenylalanine-labeled whey protein was obtained by infus-ing a Holstein cow with large quantities of L-[1-13C]phenylalanine,collecting milk, and purifying the whey protein fraction as describedpreviously (21). The whey protein fraction consisted of 93% nativewhey protein, 7% casein, and denatured whey protein and did notprovide other sources of nutrients, i.e., during processing, milk fat andlactose were removed to �1% of original milk content. The L-[1-13C]phenylalanine enrichment of labeled whey protein, which wasassessed by gas chromatography-mass spectrometry after hydrolysis,was 31.3 mole percent excess (MPE). The labeled protein met allchemical and bacteriological specifications for human consumption.

Plasma analyses. Plasma glucose (Uni Kit III, 07367204; Roche,Basel, Switzerland) concentrations were analyzed with the COBAS-FARA semiautomatic analyzer (Roche). Insulin was analyzed byradioimmunoassay (Insulin RIA kit; LINCO Research, St. Charles,

MO). Plasma (100 �l) for amino acid analyses was deproteinized onice with 10 mg dry 5-sulfosalicylic acid and mixed, and the clearsupernatant was collected after centrifugation. Plasma amino acidconcentrations were determined by HPLC, after precolumn derivati-zation with o-phthaldialdehyde (25). For plasma phenylalanine andtyrosine enrichment measurements, plasma phenylalanine and ty-rosine were derivatized to their t-butyldimethylsilyl derivatives, andtheir 13C and 2H enrichments were determined by electron ionizationGC-MS (Agilent 6890N GC/5973N; MSD, Little Falls, DE) usingselected ion monitoring of masses 336, 337, and 341 for unlabeled andlabeled (1-13C and ring-2H5) phenylalanine and masses 466, 467, 468,and 470 for unlabeled and labeled (1-13C, ring-2H2, and ring-2H4)tyrosine (27). Standard regression curves were applied in allisotopic enrichment analyses to assess linearity of the mass spec-trometer and to control for the loss of tracer. Phenylalanine andtyrosine enrichments were corrected for the presence of both the13C and 2H isotopes (4).

Muscle tissue analyses. For measurement of L-[1-13C]phenylala-nine and L-[ring-2H5]phenylalanine enrichment in mixed-muscle pro-tein, 55 mg of wet muscle was freeze-dried. Collagen, blood, andother nonmuscle fiber material were removed from the muscle fibersunder a light microscope. The isolated muscle fiber mass (10–15 mg)was weighed, and 8 vol (8 � dry wt of isolated muscle fibers �wet-to-dry ratio) ice-cold 2% perchloric acid (PCA) were added. Thetissue was then homogenized and centrifuged. The protein pellet waswashed with three additional 1.5-ml washes of 2% PCA, dried, andhydrolyzed in 6 M HCl at 120°C for 15–18 h. The hydrolyzed proteinfraction was dried under a nitrogen stream while heated to 120°C, andthen 50% acetic acid solution was added and the hydrolyzed proteinwas passed over a Dowex exchange resin (AG 50W-X8, 100–200mesh hydrogen form; Bio-Rad, Hercules, CA) using 2 M NH4OH.The eluate was divided over two vials for separate measurement ofboth L-[1-13C]phenylalanine and L-[ring-2H5]phenylalanine enrich-ment in mixed-muscle protein as described previously (14). In short,L-[1-13C]phenylalanine and L-[ring-2H5]phenylalanine were deriva-tized to their N(O,S)-ethoxycarbonyl ethyl esters and MTBSTFA-phenylethylamines, respectively (13). Thereafter, the ratios labeled/unlabeled derivatives were determined by GC-C-IRMS (FinniganMAT 252, Bremen, Germany) and GC-MS, respectively. Standardregression curves were applied to assess linearity of the mass spec-trometer and to control for loss of tracer.

Calculations. Ingestion of L-[1-13C]phenylalanine-labeled protein,intravenous infusion of L-[ring-2H5]phenylalanine and L-[ring-2H2]tyro-sine, and arterialized blood sampling were used to assess whole bodyprotein metabolism in non-steady-state conditions. Total, exogenous,and endogenous phenylalanine rate of appearance (Ra) and plasmaavailability of dietary phenylalanine (i.e., fraction of dietary phenyl-alanine that appeared in the systemic circulation, Pheplasma) werecalculated using modified Steele’s equations (6, 9). These parameterswere calculated as follows:

Total Ra �F � pV · C(t) · dEiv ⁄ dt

Eiv(t)(1)

Exo Ra �Total Ra · Epo (t) � pV · dEpo ⁄ dt

Eprot(2)

Endo Ra � Total Ra � Exo Ra � F (3)

Pheplasma � �AUCExoRa

PheProt� · BW · 100 (4)

where F is the intravenous tracer infusion rate (�mol·kg�1·min�1),pV (0.125) is the distribution volume for phenylalanine (6). C(t) is themean plasma phenylalanine concentration between two time points.dEiv/dt represents the time-dependent variations of plasma phenylal-anine enrichment derived from the intravenous tracer, and Eiv(t) is themean plasma phenylalanine enrichment from the intravenous tracer

Table 1. Subject characteristics

Whey Protein, g

10 20 35

Age, yr 73 � 2 73 � 2 73 � 1Weight, kg 77.2 � 1.6 78.0 � 1.7 77.2 � 1.3BMI, kg/m2 25.2 � 0.5 25.6 � 0.5 25.4 � 0.6Fat, % 21.1 � 0.9 21.1 � 1.0 20.8 � 1.3Lean body mass, kg 60.9 � 1.3 61.4 � 1.0 61.1 � 1.2HbA1c, % 5.7 � 0.1 5.9 � 0.1 5.6 � 0.1Basal glucose, mmol/l 5.5 � 0.1 5.6 � 0.1 5.5 � 0.1Basal insulin, mU/l 17.1 � 1.6 19.1 � 2.8 16.3 � 1.1

Values are expressed as means � SE; n � 11 subjects in each group. BMI,body mass index; HbA1c, hemoglobin A1c. Data were analyzed with ANOVA.No differences were observed between groups.

E993WHEY PROTEIN AND MUSCLE PROTEIN ACCRETION

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between two consecutive time points. Exo Ra represents the plasmaentry rate of dietary phenylalanine, Epo(t) is the mean plasma phenyl-alanine enrichment for the oral tracer, dEpo/dt represents the time-dependent variations of plasma phenylalanine (Pheplasma) enrichmentderived from the oral tracer, and Eprot is the L-[1-13C]phenylalanineenrichment in the dietary protein. Endo Ra is endogenous phenylala-nine appearance rate. PheProt is the amount of dietary phenylalanineingested, and AUCExoRa represents the area under the curve (AUC) ofExo Ra, which corresponds to the amount of dietary phenylalaninethat appeared in the blood over the 4-h period following drink intake.BW represents the subjects’ body weight in kilograms.

Total rate of disappearance of phenylalanine (Rd) equals the rate ofphenylalanine (PHE)-to-tyrosine (TYR) conversion (first step in phe-nylalanine oxidation) and utilization for protein synthesis. Theseparameters can be calculated as follows:

Total Rd � Total Ra � pV ·dC

dt(5)

PHE-to-TYR conversion � Tyr Ra ·Et(t)

Ep(t)·

Phe Rd

(Fp � Phe Rd)(6)

Protein synthesis � Total Rd � Phe hydroxylation (7)

Phe net balance � Protein synthesis � Endo Ra (8)

where Phe Rd and Tyr Ra are the flux rates for phenylalanine andtyrosine, respectively, Et(t) and Ep(t) are the mean plasma enrich-ments of L-[ring-2H4]tyrosine and L-[ring-2H5]phenylalanine, respec-tively, and Fp is the infusion rate of the phenylalanine tracer.

The fractional rate of mixed-muscle protein synthesis [fractionalsynthetic rate (FSR)] was calculated in percent per hour by theprecursor-product method (27):

FSR ��Ep

Eprecursor · t· 100 (9)

where �Ep is the delta increment of muscle protein-bound L-[ring-2H5]phenylalanine during the incorporation period. Eprecursor is theaverage plasma L-[ring-2H5]phenylalanine enrichment during the time

period for determination of amino acid incorporation, and t indicatesthe time interval (h) between biopsies.

Statistics. All data are expressed as means � SE. A two-wayrepeated-measures ANOVA with time and treatment as factors wasused to compare differences between treatments over time. In case ofa significant interaction between time and treatment, a Bonferroni posthoc test was applied to locate these differences. For non-time-depen-dent variables, one-way ANOVA with treatment as factor was used tocompare differences between treatments. Statistical significance wasset at P � 0.05. All calculations were performed using the SPSS15.0.1.1 software package.

RESULTS

Plasma analyses. Plasma insulin concentrations showed arapid, but short-lived, increase following whey protein inges-tion in all groups (Fig. 1A). Peak plasma insulin concentrationswere higher following ingestion of 35 g compared with 10 and20 g whey protein (P � 0.05). Plasma glucose concentrationsdid not change over time or between treatments and averaged5.2 � 0.1, 5.1 � 0.1, and 5.1 � 0.1 mmol/l in the 10-, 20-, and35-g experiment, respectively (Fig. 1B).

Plasma phenylalanine, leucine, and essential amino acid(EAA) concentrations over time are illustrated in Fig. 2.Following whey protein ingestion, a rapid increase in plasmaEAA concentrations was observed in all groups, with thelowest and highest concentrations following ingestion of 10and 35 g whey protein, respectively (P � 0.01). Plasmaphenylalanine concentrations were significantly higher follow-ing ingestion of 20 and 35 g compared with the ingestion of 10g whey protein (P � 0.05). The lowest and highest (peak)plasma leucine concentrations were observed in the 10- and35-g experiment, respectively (P � 0.01).

The time course of plasma L-[1-13C]phenylalanine andL-[ring-2H5]phenylalanine enrichments is illustrated in Fig. 3.Plasma L-[1-13C]phenylalanine enrichments (ingested tracer)

Fig. 1. Plasma insulin (A; mU/l) and glucose (B; mmol/l) concentrations following ingestion of 10 (n � 11), 20 (n � 11), and 35 (n � 11) g whey protein. Valuesrepresent means � SE. Data were analyzed with repeated-measures ANOVA (time � treatment). In case of a significant interaction, a Bonferroni post hoc testwas applied to locate these differences. Time effect, P � 0.001; treatment effect, P � 0.05; time � treatment interaction, P � 0.01. *35 g significantly highercompared with 10 g, P � 0.05.

E994 WHEY PROTEIN AND MUSCLE PROTEIN ACCRETION


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rapidly increased after ingestion of the test drinks, with thelowest and highest enrichments following ingestion of 10 and35 g whey protein, respectively (P � 0.01). The plasmaL-[ring-2H5]phenylalanine enrichment (infused tracer) de-creased following whey protein ingestion in all groups, withlower values observed during the early stages following inges-tion of 20 and 35 g compared with 10 g whey protein (P �0.05). Following this initial decrease, plasma L-[ring-2H5]pheny-lalanine enrichments slowly returned to baseline levels, with

higher values following ingestion of 10 g compared with 20and 35 g whey protein (P � 0.05).

Whole body protein metabolism. Whole body protein me-tabolism over time is presented in Fig. 4. Ingestion of thelabeled whey protein resulted in a rapid rise in exogenousphenylalanine appearance rate (Fig. 4A), with the lowest andhighest (peak) values observed following ingestion of 10 and35 g whey protein, respectively (P � 0.05). Total exogenousphenylalanine appearance, expressed as AUC over 4 h, was

Fig. 2. Plasma phenylalanine (A), leucine (B), and essential amino acid (EAA; C) concentrations (�mol/l) following ingestion of 10 (n � 11), 20 (n � 11), and35 (n � 11) g whey protein. Values represent means � SE. Data were analyzed with repeated-measures ANOVA (time � treatment). In case of a significantinteraction, a Bonferroni post hoc test was applied to locate these differences. AA, amino acid. Plasma EAA concentrations: time effect, P � 0.001; treatmenteffect, P � 0.001; time � treatment interaction, P � 0.001. Plasma phenylalanine concentrations: time effect, P � 0.001; treatment effect, P � 0.01; time �treatment interaction, P � 0.001. Plasma leucine concentrations: time effect, P � 0.001; treatment effect, P � 0.001; time � treatment interaction, P � 0.001.*35 g significantly different compared with 10 g, P � 0.05; $35 g significantly different compared with 20 g, P � 0.05; #20 g significantly different comparedwith 10 g, P � 0.05.

Fig. 3. Plasma L-[1-13C]phenylalanine (A) and L-[ring-2H5]phenylalanine (B) enrichments expressed as molar percent excess (MPE) following ingestion of 10(n � 11), 20 (n � 11), and 35 (n � 11) g whey protein. Values represent means � SE. Data were analyzed with repeated-measures ANOVA (time � treatment).In case of a significant interaction, a Bonferroni post hoc test was applied to locate these differences. Plasma L-[1-13C]phenylalanine enrichments: time effect,P � 0.001; treatment effect, P � 0.001; time � treatment interaction, P � 0.001. Plasma L-[ring-2H5]phenylalanine enrichments: time effect, P � 0.001;treatment effect, P � 0.001; time � treatment interaction, P � 0.001. *35 g significantly different compared with 10 g, P � 0.05; $35 g significantly differentcompared with 20 g, P � 0.05; #20 g significantly different compared with 10 g, P � 0.05.



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calculated as a fraction of the total amount of phenylalaninethat was ingested (Eq. 4). The fraction of dietary phenylalaninethat appeared in the systemic circulation during the 4-h post-prandial period was 61 � 1, 63 � 3, and 59 � 2% followingingestion of 10, 20, and 35 g whey protein and did not differamong treatments. Endogenous phenylalanine appearance ratesdecreased following whey protein ingestion and did not differamong treatments, although a trend (P � 0.08) toward lowervalues following 35 g compared with 10 g was observed at t �180–240 min (Fig. 4B). Total phenylalanine disappearancerate (Fig. 4C) equals the rate of phenylalanine-to-tyrosineconversion, which is the first step in phenylalanine oxidation(Fig. 4D), and the utilization for protein synthesis. Phenylala-

nine-to-tyrosine conversion rates directly increased followingwhey protein ingestion in all treatments and returned to base-line values by the end of the 4-h period. The highest amount(35 g) showed greater values at t � 45–120 and t � 90–120min compared with the ingestion of 10 and 20 g whey protein(P � 0.01), respectively.

Whole body protein metabolism expressed as AUC in thebasal period and postprandial period is presented in Fig. 5.Phenylalanine released into the circulation from whole bodyprotein breakdown, expressed as AUC of Endo Ra, decreasedin all groups to the same extent following protein ingestioncompared with basal values (P � 0.01). Phenylalanine used forwhole body protein synthesis, expressed as AUC of total Rd

Fig. 4. Whole body phenylalanine (PHE) kinetics over time following ingestion of 10 (n � 11), 20 (n � 11), and 35 (n � 11) g whey protein (�molphenylalanine·kg�1·min�1). Exogenous phenylalanine appearance rates (Ra) (A), endogenous phenylalanine appearance rates (B), total phenylalanine disap-pearance rates (C), and phenylalanine-to-tyrosine conversion rates (D). Values represent means � SE and are expressed as �mol·kg�1·min�1. Data were analyzedwith repeated-measures ANOVA (time � treatment). In case of a significant interaction, a Bonferroni post hoc test was applied to locate these differences.Exogenous phenylalanine appearance rates: time effect, P � 0.001; treatment effect, P � 0.001; time � treatment interaction, P � 0.001. Endogenousphenylalanine appearance rates: time effect, P � 0.001; treatment effect, P � 0.34; time � treatment interaction, P � 0.05. Total phenylalanine disappearancerates: time effect, P � 0.001; treatment effect, P � 0.001; time � treatment interaction, P � 0.001. Phenylalanine-to-tyrosine conversion rates: time effect,P � 0.001; treatment effect, P � 0.001; time � treatment interaction, P � 0.001. *35 g significantly different compared with 10 g, P � 0.05; $35 g significantlydifferent compared with 20 g, P � 0.05; #20 g significantly different compared with 10 g, P � 0.05.



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minus phenylalanine-to-tyrosine conversion rate, increased inall groups following protein ingestion compared with basalvalues (P � 0.05). Whole body protein synthesis was higherfollowing ingestion of 20 and 35 g compared with 10 g wheyprotein (P � 0.01). Protein oxidation, expressed as AUC ofphenylalanine-to-tyrosine conversion rate, increased in allgroups following protein ingestion compared with basal values(P � 0.01). Protein oxidation was higher following ingestionof 35 g compared with 10 g (P � 0.01). Whole body netprotein balance increased in all groups following protein in-gestion compared with basal values (P � 0.01), with the lowestand highest values observed following ingestion of 10 and 35g, respectively (P � 0.01).

Muscle tracer analysis. The increment in muscle protein-bound L-[ring-2H5]phenylalanine enrichment during the basalperiod (between the first and the second biopsy) was 0.0059 �0.0005, 0.0059 � 0.0005, and 0.0065 � 0.0006 MPE in thegroups that ingested 10, 20, and 35 g whey protein, respec-tively, and did not differ among treatments. The increment inmuscle protein-bound L-[ring-2H5]phenylalanine enrichmentsin the postprandial period (between the second and thirdbiopsy) was 0.0102 � 0.0010, 0.0119 � 0.0012, and 0.0136 �0.0009 MPE in the 10-, 20-, and 35-g experiment, respectively(P � 0.08). The increment in muscle protein-bound L-[1-13C]phenylalanine enrichments in the postprandial period dif-fered substantially among experiments (P � 0.01; Fig. 6) andwas higher following ingestion of 35 g compared with 10 (P �0.01) or 20 (P � 0.05) g whey protein.

Mixed-muscle protein synthesis rates. Mixed-muscle proteinsynthesis rates are expressed as FSR and presented in Table 2.In the basal period, FSR values did not differ among treat-ments. In contrast, FSR values were significantly differentamong treatments in the postprandial period (P � 0.01).Ingestion of 35 g resulted in significantly higher FSR valueswhen compared with basal values (P � 0.05) or ingestion of 10g whey protein (P � 0.05).

DISCUSSION

In the present study, intrinsically L-[1-13C]phenylalanine-labeled whey protein was used to assess digestion and absorp-tion kinetics of whey protein and its subsequent incorporationin newly synthesized muscle protein in vivo in older males.The present study shows that ingestion of 35 g whey proteinresults in greater amino acid absorption and subsequent stim-ulation of de novo muscle protein synthesis compared with theingestion of 10 or 20 g whey protein in healthy, older men.

Whey protein ingestion has been shown to effectively pro-mote postprandial muscle protein accretion in older adults (19).However, information on the impact of the amount of wheyprotein ingested by older adults on protein digestion, aminoacid absorption, and postprandial muscle protein accretion hasnot been established. Following the ingestion of 10, 20, and 35g intrinsically L-[1-13C]phenylalanine-labeled whey protein, arapid increase in plasma insulin concentrations (Fig. 1A),plasma amino acid concentrations (Fig. 2), and plasma L-[1-13C]phenylalanine enrichments (Fig. 3A) was found, with thelowest and highest values observed following ingestion of 10and 35 g, respectively. By combining plasma phenylalanineconcentrations and tracer enrichments, the fraction of dietaryphenylalanine that appeared in the circulation was calculated(Eq. 4). These fractions were 61 � 1, 63 � 3, and 59 � 2% in

Fig. 5. Whole body protein metabolism expressed as area under the curve(�mol phenylalanine/kg) following ingestion of 10 (n � 11), 20 (n � 11), and35 (n � 11) g whey protein. Values represent means � SE. Data were analyzedwith ANOVA with Bonferroni correction. *35 g significantly different com-pared with 10 g, P � 0.05; $35 g significantly different compared with 20 g,P � 0.05; #20 g significantly different compared with 10 g, P � 0.05;‡significantly different compared with baseline (0 g).

Fig. 6. Muscle protein-bound L-[1-13C]phenylalanine enrichments (MPE) fol-lowing ingestion of 10 (n � 11), 20 (n � 11), and 35 (n � 11) g whey protein.Values represent means � SE. Data were analyzed with ANOVA withBonferroni correction. *35 g significantly different compared with 10 g, P �0.01; $35 g significantly different compared with 20 g, P � 0.05

Table 2. Mixed-muscle protein FSR

Whey Protein, g

10 20 35

Basal FSR, %/h 0.035 � 0.002 0.037 � 0.003 0.039 � 0.003Postprandial FSR, %/h 0.029 � 0.004 0.041 � 0.004 0.052 � 0.004*‡Difference from basal, % �1 � 14 16 � 13 44 � 16*

Values are expressed as means � SE; n � 11 subjects in each group.Mixed-muscle protein fractional synthetic rates (FSR) in the fasted, basalperiod and following ingestion of 10, 20, and 35 g whey protein. Data wereanalyzed with ANOVA. *35 g significantly different compared with 10 g, P �0.05. ‡Significantly different compared with basal, P � 0.05.



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the groups that received 10, 20, and 35 g whey protein,respectively, and did not differ among treatments. Previouswork has suggested that first-pass clearance of amino acids byvisceral tissues increases with increasing amounts of proteinconsumed (26). In agreement, when expressed as absoluteamounts, 4 � 1, 8 � 1, and 14 � 1 g whey protein-derivedamino acids were retained in the gut and did not appear in thecirculation during the 4-h postprandial period after ingesting10, 20, and 35 g whey protein, respectively. Despite greaterretention in the gut, more amino acids became available in thecirculation following ingestion of 35 g whey protein comparedwith the ingestion of 10 or 20 g whey protein in older men.

Exogenous phenylalanine rates of appearance increased rap-idly following whey protein ingestion in all groups, with peakvalues being reached within 30–60 min (Fig. 4A). No othermacronutrients were included because we aimed to assess thepostprandial response of different amounts of whey proteinwithout possible confounding effects of carbohydrate and/orfat intake on protein digestion and absorption kinetics (10, 17).Consequently, nonisocaloric protein drinks were compared,and, as a result, different insulin responses were observedbetween experiments (Fig. 1A). Differences in the insulinresponse are unlikely to have modulated the observed muscleprotein synthetic response because increases in plasma EAAconcentrations, and not insulin per se, are responsible forstimulating postprandial muscle protein synthesis (5, 16). Cir-culating insulin levels are regarded permissive rather thanmodulatory with concentrations of �10–15 mU/l being re-quired to allow a maximal muscle protein synthetic response(5, 7, 11, 16). In the present study, plasma insulin levelsexceeded these concentrations in all treatments (Fig. 1A). Inagreement, the observed plasma insulin levels were also suf-ficient to maximize the inhibition of protein breakdown in alltreatments (Fig. 5).

Muscle protein FSR following whey protein ingestion werecalculated under non-steady-state conditions, which may un-derestimate (peak) postprandial muscle protein synthesis rates(Table 2). Because we were more interested in an aggregated4-h postprandial response, we also assessed the metabolic fateof whey protein-derived amino acids in de novo muscle protein(Fig. 6). Only ingestion of 35 g whey protein significantlyincreased muscle protein synthesis rates compared with base-line values. The latter seems to be at odds with the whole bodykinetics (Fig. 5), showing a positive whole body protein bal-ance in all treatments. However, whole body protein kineticsdo not necessarily represent skeletal muscle tissue, sincesplanchnic tissues and other organs contribute largely to post-prandial protein metabolism (26).

The present study shows that ingestion of 35 g whey proteinresults in greater amino acid absorption and subsequent use forde novo muscle protein synthesis compared with the ingestionof 10 or 20 g whey protein. With habitual protein ingestion ina single meal varying between 10 g (breakfast) and 35 g(dinner) in institutionalized and independently living elderly(24), it has been suggested that increasing the amount ofprotein at breakfast and/or lunch may represent an effectivedietary strategy to stimulate postprandial muscle protein accre-tion and, as such, improve muscle mass preservation in olderadults. It should be noted that the dose-response relationship islikely specific for more rapidly digestible protein sources.Previous work from our laboratory suggests that ingesting

greater amounts of more slowly digestible protein does notresult in greater amino acid absorption (14, 19). In agreement,we observed a dose-response effect between the amount ofprotein ingested and subsequent amino acid absorption ratesfollowing ingestion of a casein hydrolysate but not intactcasein. Besides protein digestion and absorption kinetics,amino acid composition as well as coingestion of other ma-cronutrients are likely to modulate the muscle protein syntheticresponse following meal ingestion. Nonetheless, the presentedwork clearly underlines the impact of the amount of proteiningested on subsequent amino acid availability and muscleprotein synthesis.

In conclusion, the ingestion of 35 g whey protein results inmore amino acids being absorbed and subsequently used for denovo muscle protein synthesis compared with the ingestion of10 or 20 g whey protein. These observations imply thatanabolic resistance to food intake in older adults can, at leastpartly, be compensated for by ingesting a greater amount ofwhey protein.

ACKNOWLEDGMENTS

We acknowledge the assistance of Alexandra Kiskini and Zouhair Ariss forskillful technical assistance and the enthusiastic support of the subjects whovolunteered to participate in these experiments.

DISCLOSURES

This trial was registered at clinicaltrials.gov as NCT00557388 and was notfunded by external sources.

AUTHOR CONTRIBUTIONS

Author contributions: B.P. and L.J.v.L. conception and design of research;B.P., B.B.G., A.d.L., and A.H.Z. performed experiments; B.P., A.P.G., andJ.M.S. analyzed data; B.P. and L.J.v.L. interpreted results of experiments; B.P.prepared figures; B.P. drafted manuscript; B.P. and L.J.v.L. edited and revisedmanuscript; B.P., B.B.G., and L.J.v.L. approved final version of manuscript.

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Amino acid absorption and subsequent muscle …...Amino acid absorption and subsequent muscle protein accretion following graded intakes of whey protein in elderly men Bart Pennings,1,2