Nutrient Metabolism

Evaluation of the Arteriovenous Difference Techniqueto Simultaneously Estimate Protein Synthesis andDegradation in the Hindlimb of Fed and Chronicallyunderfed Steers1'2

YVES R. BOISCLA1R,3 ALAN W. BELL, FRANK R. DUNSHEA,4MARIE HARKINS A/YDDALE E. BAUMAN5

Department of Animal Science, Cornell university, Ithaca, NY 14853

ABSTRACT Our objectives were to validate a technique to measure protein turnover based on net fluxes oftyrosine radioactivity and mass across the hindlimb, andto describe the impact of nutrition on hindlimb proteinkinetics and nutrient utilization in growing Holsteinsteers. External iliac vessels were catheterized to allowfor measurements of hindlimb arteriovenous (AV) differences and blood flow. Animals were used in a singlereversal design with 16-d periods. Treatments wereeither 60% (0.6M) or 220% (2.2M) of the energy required for maintenance. On d 12 and 14 of each period,a primed-continuous infusion of L-[side chain-2,3-^H]tyrosine was initiated, followed by a 4-h sampling period to assess hindlimb nutrient utilization andprotein kinetics. Well-fed steers exhibited rates ofhindlimb utilization for non-nitrogenous energy substrates and amino acids that were consistent with valuespreviously reported for well-fed ruminants. Underfeedingdecreased hindlimb blood flow (62%), oxygen consumption (51%) and utilization of respiratory fuels(64-73%). Protein synthesis was also reduced (49%),leading to net nitrogen loss from the hindlimb. The estimated energy cost of protein synthesis accounted for13% of hindlimb oxygen consumption across intakelevels. Hindlimb protein balance was consistent withwhole-body balance, adding credence to derived rates ofdegradation. Overall, data support the validity of the AVdifference technique to estimate components of hindlimbprotein turnover. J. Nutr. 123: 1076-1088, 1993.

INDEXINGKEYWORDS:

•hindlimb •protein turnover•arteriooenous difference technique

undernutrition cattle

Understanding the regulation of protein accretionand the primary mechanisms of any modifyingtreatment requires a simultaneous assessment of bothprotein synthesis and degradation. Traditionally,protein synthesis has been estimated in vivo by measuring labeled amino acid incorporation into tissue

protein after either continuous infusion or injectionof a flooding dose (reviewed by Reeds 1992). Thisapproach suffers from two major drawbacks. First, itusually requires slaughter of the animal for tissuesampling, negating repeated measurements on thesame animal. Second, label incorporation techniquesdo not allow for a simultaneous determination ofprotein degradation. These rates can be estimatedonly by relating protein synthesis to balance, andsignificant errors are incurred because differentanimals, time periods and assumptions are involved(reviewed by Reeds 1992).

An alternative approach has been reported to allowsimultaneous and repeated measurement of rates ofprotein synthesis, accretion and degradation inmuscle (Barrett et al. 1987, Oddy and Lindsay 1986,Pell et al. 1986). The method is a simple extension ofthe traditional arteriovenous (AV)6 difference tech

nique used to study organ metabolism. Rates of

1Presented in part at the 72nd Annual Meeting of the Federation

of the American Societies for Experimental Biology, April 1988, LasVegas, NV, [Boisclair, Y. R., Bauman, D. E., Bell, A. W. & Dunshea,F. R. (1988| Muscle protein synthesis and whole body N retentionin fed and underfed steers. FASEB J. 2: A848 (abs.)].

^Supported in part by Cornell University Agricultural Experiment Station and USDA grant 85-CRCR-1-1833.

3Recipient of scholarships from the Natural Sciences and Re

search Council of Canada and Purina Mills, Inc. Current address:Molecular, Cellular and Nutritional Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases,National Institutes of Health, Building 10, Room 8D14, Bethesda,MD 20892.

4Current address: Animal Research Institute, Department of

Agriculture and Rural Affairs, Werribee VIC 3030, Australia.5To whom correspondence and reprint requests should be ad

dressed.Abbreviations used: AV, arteriovenous; IGF-I, insulin-like

growth factor I; NEFA, nonesterified fatty acids; PAH, p-aminohip-puric acid.

0022-3166/93 $3.00 ©1993 American Institute of Nutrition. Received 24 July 1992. Accepted 13 January 1993.

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PROTEIN TURNOVER IN FED AND UNDERFED STEERS 1077

protein synthesis and accretion are inferred from thedisappearance of tracer and tracéeamino acid across adefined muscle mass (or organ), and the rate of proteindegradation is obtained by difference. Thus, this technique theoretically eliminates or minimizes limitations described above for the label incorporationmethod.

Comparable estimates of protein synthesis havebeen obtained by the AV and label incorporationmethods (Gelfand and Barrett 1987, Oddy and Lindsay1986). However, demonstration that the rate ofprotein accretion is quantitatively consistent withwhole-body nitrogen balance is still lacking. Suchvalidation is crucial for the rate of protein degradation, which is calculated as the difference betweenprotein synthesis and protein accretion, to have anysignificance. Furthermore, it is not readily obviousthat the AV method will faithfully describe proteinkinetics in the hindlimb of conscious animals, particularly when considering that variables measured(blood flow, AV differences for radioactivity and mass)may not be maintained in steady state as easily as inanesthetized animals (Barrett et al. 1987) or cooperative subjects such as humans (Fryburg et al. 1990,Gelfand and Barrett 1987). Therefore, the major objective of the present study was to evaluate the abilityof the AV difference technique to adequately describechanges in hindlimb protein turnover of steers undergoing periods of negative and positive N balance.Additionally, the impact of nutrition on the relationship of energy expenditure, nutrient utilizationand protein turnover of skeletal muscle in ruminantshas been studied mostly in mature sheep (Pell et al.1986, Pethick et al. 1984, Teleni et al. 1986). Thus, asecond objective of the present study was to describesimultaneously the impact of nutrition on utilizationof energy-yielding nutrients and protein turnover inyoung cattle with rapid growth potential.

While this paper was in press, similar data forsheep were published by Harris et al. (1992).

MATERIALS AMD METHODS

Animals and design. This study was conductedwith the approval of the Cornell University Institutional Animal Care and Use Committee. Four Hoi-stein steers (230-250 kg body wt, ~7 mo old) wereprepared for measurements of AV differences andblood flow across a defined muscle mass using surgical procedures as previously described (Boisclair etal. 1993). Briefly, the in situ preparation consisted ofcatheters in the external iliac arterial and venousvessels of one leg and an arterial catheter in thecontralateral leg. This preparation contains -62%skeletal muscle by weight (Boisclair et al. 1993).

Animals were given 7 to 10 d to recover fromsurgery. They were then used in a single reversal

design with 16-d periods in which treatments consisted of two levels of intake. Steers were fed a complete mixed diet (Table 1) in amounts representingeither 60% (0.6M) or 220% (2.2M) of the predicteddaily energy requirement for maintenance (NRC1984). Feeding was hourly to approximate steady-

state conditions for digestion and metabolism. At the2.2M level, soybean meal (Table 1) was added to theration (-7% dry matter intake) to provide a 15%excess of the predicted crude protein requirement.

Nitrogen balance. On d 7 of treatment, steers werefitted with a continuous urine collection system. Thecollection system consisted of a rubber funnel attached to the animal and connected to a collectionjug. A pump generated a continuous vacuum andensured complete urine collection. Total collection offeces and urine occurred on d 9-16 of each treatmentperiod. Feed, urine and feces were weighed, mixed,subsampled, and stored daily at 4°C.Preservation of

urine and trapping of ammonia were achieved byaddition of sulfuric acid (8 mol/L), in volume sufficient to maintain a pH <2.00, to the collecting jug atthe start of each day. Feces were preserved by addingformaldehyde (37% solution, 1 mL/kg wet feces) atthe time of mixing. On the last day of the collectionperiod, subsamples were combined and mixed. Feedand feces dry matter were determined by drying to aconstant weight in a forced draft oven at 60°C.

Nitrogen content was determined by the macro-Kjeldahl method (AOAC 1984).

Hindlimb metabolism. A single solution of L-[sidechain-2,3-3H]tyrosine (Amersham, Arlington Heights,

IL) was prepared and used in two series of measurements per treatment period on each steer. Final activity of the infúsate (999.0 MBq/L) was obtained bymixing exact volumes of sterile saline andultrafiltered stock solution of [3H]tyrosine (36.7 GBq/

L). On d 12 and 14 of each period, a primed (12.3MBq), continuous infusion of [3H]tyrosine (299.7 KBq/

min) commenced at 1115 h and continued for 5 h.Infúsate was delivered with a syringe pump (model SE400, Vial Medical, Grenoble, France) into a jugularvein previously catheterized on d 7.

Hindlimb blood flow was determined by measuringthe downstream dilution of a p-aminohippuric acid(PAH) solution (0.257 mol/L) infused into the externaliliac artery. The protocol consisted of a primed (6.2mmol) continuous infusion (0.309 mmol/min) using asyringe pump (model 915, Harvard Apparatus, SouthNatick, MA). The PAH infusion commenced at 1130h and continued during the isotope infusion.

Arterial blood samples (n = 3) were collected duringthe 1.5-h interval preceding the isotope infusion toassess residual levels of radioactivity on the second,third and fourth infusions. After tracer equilibration,samples were collected between 1215 and 1615 h,during which animals were standing continuously. At15 and 45 min after each hour, arterial and venous

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1078 BOISCLAIR ET AL.

TABLE 1

Composition of diet

Complete Soybeanmixed diet1meal1Ingredient,

g/kgCrackedcornChopped

hayFishmealComoilSoybean

mealMineralmix2Composition

Crude protein, g/kgMetabolizable energy,3 MJ/kg520400302014

16120

53611.3 13.2

Expressed on a dry matter basis.^Mineral mix supplement contained (on a dry matter basis):

defluorinated phosphate, 680 g/kg; trace mineralized NaCl, 320 g/kg. Trace minerals included iron, manganese, zinc, iodine, cobaltand selenium.

Estimated from tabular values for each ingredient (NRC 1984|.

blood samples were simultaneously withdrawn fromthe external iliac vessels. A portion was placed intubes and allowed to clot for 4 h at room temperature.Following overnight refrigeration (4°C),serum was

obtained and frozen at -2CTC. The remaining bloodwas immediately mixed with heparin (6 x IO4 u/L)

and kept on ice. An aliquot was centrifuged (1500 x gat 4°C, 20 min) and plasma stored at -20°C. The

remainder of the heparinized blood was subjected topreparative chemistry for later analyses (see analyticalmethods).

Blood from both vessels was also obtained at eachsampling in heparinized 3-mL syringes. Syringes weresealed, placed on ice and immediately analyzed foroxygen saturation and hemoglobin concentrationwith an automatic direct-reading photometer (he-

moximeter model OSM2, Radiometer, Copenhagen,Denmark) and for packed cell volume bymicrocapillary centrifugation.

To increase the precision of the estimate ofhindlimb blood flow, two additional samples (1.5 mL)were obtained on the hour and at 30 min after eachhour and used exclusively for PAH analysis.

Analytical methods. All blood deproteinizationswere completed within l h of sampling. Blood usedfor analyses of amino acids was frozen (-85°C, 40

min) immediately after sampling and then thawed tolyse red blood cells. Blood and ice-cold sulfosalicylicacid (500 g/L), containing the internal standardnorleucine (616 /¿mol/L), were combined (4:1) andextensively vortexed. After centrifugation (31,000 x gat 4°C,30 min), supernatant was added to tubes con

taining lithium hydroxide (0.4 /¿mol/mL)to adjust thefinal pH to 2.5-2.7 and frozen at -85°C until ana

lyzed. Amino acid content was determined by automated HPLC (model 334, Beckman Instruments, PaloAlto, CA). Amino acid separation was achieved with agradient elution system of lithium citrate buffers onan ion exchange column packed with polystyrenedivinylbenzene (lithium form). Quantification ofamino acids concentrations was by fluorimetry following post-column derivatization with or-thophthaldehyde.

For determination of [3H]tyrosine, blood and ice-

cold sulfosalicylic acid (100 g/L) were mixed (1:1) andsupernatant separated and stored as before. Separationof [3H]tyrosine from other radiolabeled metabolites

was achieved by ion exchange chromatography.Minicolumns (Isolab, Akron, OH) were filled with 0.5mL of a cation exchange resin (Dowex 50X8, 200-400mesh, Bio-Rad Laboratories, Richmond, CA)equilibrated with HCl (0.01 mol/L). A volume of sulfosalicylic acid supernatant containing a minimum of1000 cpm of pHjtyrosine was combined with 30 Bq of[U-14C]tyrosine as an internal standard and applied to

the column. Labeled products of catabolism (waterand organic acids) and amino acids were sequentiallyeluted with HCl (0.01 mol/L) and NaOH (0.8 mol/L).Preliminary experiments demonstrated that 98% ofthe radioactivity present in the NaOH fraction wasassociated with tyrosine. The NaOH effluent wasneutralized with HCl (3 mol/L), evaporated to drynessat 45°Cunder air and solubilized with HCl (0.01 mol/

L). Scintillation fluid (Aquasol-2, Du Pont NEN,Boston, MA) was added and radioactivity quantifiedusing a dual channel liquid scintillation analyzer withexternal standard correction (2200 Tri Carb, PackardInstruments, Downers Grove, IL). In addition, ali-quots of infúsate were dried under nitrogen, reconstituted with HCl (0.01 mol/L), mixed with scintillation fluid and counted. Intra- and inter-assay CV forradioactivity determination averaged less than 1.5 and2%, respectively.

Perchloric acid extracts were prepared by mixingequal volumes of blood and ice-cold perchloric acid(100 g/L). Samples were centrifuged (31,000 x g at4°C,30 min) and the supernatant neutralized with

KOH (200 g/L). After centrifugation to remove theprecipitated potassium perchlorate, the supernatantwas stored at -85°Cuntil analyzed for acetate (Guynn

and Veech 1974), 3-hydroxybutyrate (Kientsch-Engeland Siess 1985) and lactate (Gutmann and Wahlefeld1974) using specific enzymatic assays.

Trichloroacetic acid filtrates of blood were prepared and analyzed for PAH by a modification of themethod of Katz and Bergman (1969). Aliquots of bloodfor glucose analysis were frozen (-20°C)with sodium

fluoride (19 /miol/L). Filtrates were prepared bydeproteinization with zinc sulfate and bariumhydroxide, and glucose was analyzed with a commercial kit (kit #510, Sigma Chemical, St. Louis,MO). Plasma samples were analyzed by kits for

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PROTEIN TURNOVER IN FED AND UNDERFED STEERS 1079

nonesterified fatty acids (NEFA) (Wako Chemicals,Dallas, TX) and for urea (kit #640B, Sigma Chemical).Intra- and inter-assay CV for the PAH and metaboliteassays were less than 1.5 and 3%, respectively.

Specific double antibody RIA were used to assaysamples for hormones. The RIA for serum insulin-likegrowth factor I (IGF-I) was described by Flaut et al.(1991). Monoclonal antibody 3D1 prepared againsthuman IGF-I was provided by the National Instituteof Diabetes, Digestive and Kidney Diseases (NationalHormone and Pituitary Program, University ofMaryland School of Medicine, Baltimore, MD), and N-methionyl human IGF-I (lot 742-44, Pitman-Moore,Northbrook, IL) was used for iodination and standards. The range of the assay for IGF-I was 0.2 to 8 ng/tube and intra- and inter-assay CV were 4.5 and 6%,respectively. Plasma somatotropin was determinedusing antibody supplied by the National Institute ofDiabetes, Digestive and Kidney Diseases (anti-oGH-2;AFP-CO123080) and pituitary-derived bovinesomatotropin (lot 12, 1.3 lu/mg protein; Miles Laboratories, Naperville, IL) for standards and iodination(Cohick et al. 1989). Range of the somatotropin assaywas 0.2 to 8 ng/tube, and intra- and inter-assay CVwere 4.1 and 7.1%, respectively. Serum insulin RIAwas based in a guinea pig anti-porcine antibody (cat.#299-230P, Cambridge Medical Technology, Billerica,MA) using porcine insulin for standards (MicromedicSystem, Horsham, PA) and iodinated hormone (Cambridge Medical Technology). Assay range was from0.02 to 0.12 ng/tube, with an intra- and inter-assayCV of 7.0 and 6.0%, respectively.

Calculations and statistical analyses. Volumesused for deproteinization were derived from weightsrecorded to the nearest milligram and specificgravities of blood, plasma and reagents. Blood concentrations of metabolites measured on filtrates werethen obtained by applying appropriate correction.Overall means generated from a single day were usedin the calculations.

Hindlimb blood flow (L/min) was calculated bydividing the PAH infusion rate (mmol/min) by thedifference in PAH concentration (mmol/L) betweenthe external iliac vein and the contralateral artery.

The net hindlimb uptake or utilization (mmol or/¿mol/min)of nutrients was obtained as follows:

Net uptake = (CA - Cv) x Blood flow (1)

where CA and Cy are arterial and venous concentrations (mmol or /¿mol/L),respectively.

Tyrosine and phenylalanine are neither synthesizednor degraded in rat muscle (Chang and Goldberg1978). This assumption was specifically verified fortyrosine with bovine skeletal muscle using the samein vitro system (J. Peters, The Upjohn Co.,Kalamazoo, MI, personal communication). Thus, atsteady state, the net uptake of tyrosine andphenylalanine reflects muscle protein balance (mg/min) as follows:

Protein balance = (Net uptake x MW)

(FC x 1000)(2)

where MW is the molecular weight of the amino acidand FC is the fractional contribution (g/kg crudeprotein) of the amino acid to protein. The fractionalcontributions for tyrosine and phenylalanine in youngcalves are 26 and 35 g/kg carcass crude protein,respectively (Williams 1978). Values for carcass ratherthan muscle protein were used because other tissuescontribute to the external iliac preparation (Boisclairet al. 1993).

Because incorporation into protein is the onlymetabolic fate of tyrosine in muscle, the unidirectional utilization of tyrosine provides an estimate ofprotein synthesis (mg/min) as follows:

Protein synthesis(AVBq x Blood flow x MW)

(VSRA x FC x 1000) (3)

where AVgq is the arteriovenous difference of[3H]tyrosine in blood (kBq/L) and VSRAis the specificradioactivity of tyrosine (kBq//imol) in venous blood.Protein degradation (mg/min) was obtained bydifference between protein synthesis and proteinbalance.

Estimates of hindlimb energy expenditure and theenergy cost of protein synthesis were expressed inATP equivalents by assuming 1) a minimum cost of 5mol of ATP per mole of amino acid incorporated, 2)an average molecular weight of 110 for amino acidsand 3) a yield of 6 mol of ATP for each mole ofoxygen consumed (Reeds et al. 1985). Substrate/oxygen quotients were calculated using 6, 2 and 4.5mol of oxygen consumed per mole of glucose, acetateand 3-hydroxybutyrate taken up by the hindlimb,respectively, and assuming complete oxidation.

The apparent whole-body turnover rate (mmol/min) of tyrosine was calculated from the rate of tracerinfusion (KBq/min) divided by the steady-state arterialspecific radioactivity. The specific radioactivity wascorrected for residual levels of activity observed at thestart of the infusion (always <4% of steady-statelevels of radioactivity).

Because there were no day effects for any variable(P >0.20), measurements taken on two different dayswithin a nutritional treatment period were averaged.The statistical model accounted for animal, treatmentperiod and treatment. Analyses were by the methodof least squares, using the general linear modelprocedure of SAS (1985).

RESULTS

Whole-body metabolism. Effects of nutritionaltreatment on whole-body N metabolism are reported

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1080 BOISCLAIR ET AL.

in Table 2. The 2.2M level of intake resulted in a rateof N accretion considered normal for Holstein steersof this age (M. L. Thonney, Cornell University,Ithaca, NY, personal communication). Lower Nintake at the 0.6M level was associated with reducedexcretion of fecal and urinary N, resulting in a net Nloss of 9.3 g/d (P < 0.005). Body weight changesparalleled the N balance data (-2.2 and 1.2 kg/d for0.6M and 2.2M, P < 0.01). Digestibility of dry matterimproved during underfeeding (P < 0.02), probablyrelated to the increased residence time of feeds in thedigestive tract.

Effects of treatment on metabolites and hormonesare listed in Tables 3 and 4. Intake below maintenance reduced arterial blood concentrations ofglucose, acetate, 3-hydroxybutyrate, lactate andplasma urea, and quadrupled plasma NEFA concentrations (Table 3). Underfeeding also increased bloodoxygen concentration, due entirely to hemoconcen-

tration as shown by elevated hemoglobin concentration and packed cell volume. Circulating concentrations of insulin and IGF-I were decreased withunderfeeding, but the concentration of somatotropinwas unaltered. Arterial levels of most amino acidswere also reduced when animals were underfed (Table4). This was particularly evident for asparagine,glutamine, alanine, isoleucine, leucine, tyrosine,phenylalanine and arginine. In contrast, levels oflysine and glutamate increased with underfeeding.

Hindlimb metabolism. Blood flow, AV differencesand net uptake of respiratory fuels across thehindlimb are summarized in Table 5. Chronic underfeeding decreased blood flow by 62% (P < 0.001), achange partially compensated by a 36% increase inthe oxygen AV difference (P < 0.05). Underfeedingdiminished the acetate AV difference and caused aswitch from net uptake to net export of lactate. Aftercorrection for lactate net exchange, glucose AV

difference (0.094 vs. 0.177 mmol/L) was less duringthe period of underfeeding (P < 0.05). Overall, changesin hindlimb blood flow and AV differences resulted insignificant reductions in net utilization for glucose,acetate, 3-hydroxybutyrate, lactate and oxygen duringthe period of underfeeding.

The hindlimb AV difference and net uptake ofamino acids are presented in Table 6. Taurine andcitrulline AV differences during a sampling session(mean of nine AV differences) were not consistentlydifferent from zero (P > 0.10) at either level of intakeand consequently are not listed. Well-fed animals exhibited positive AV differences and net uptake of allamino acids except glutamine. Chronic under-nutrition greatly increased the negative AV differencefor glutamine and resulted in a shift from positive tonegative AV differences for threonine, glycine,alanine, tyrosine and phenylalanine. Associated nethindlimb uptakes were thus clearly negative andlower than those observed at the higher intake level(P < 0.02, except for glycine, P < 0.09). Other aminoacid AV differences remained positive (serine,glutamate and ornithine) or not consistently differentfrom zero (asparagine, valine, isoleucine, leucine,histidine, lysine and arginine) during underfeeding.Nevertheless, their net fluxes were considerablydiminished compared with the period of normalfeeding (Table 6).

Hindlimb tyrosine and protein kinetics. PlateauSRA was always achieved within the first 90 min of[3H]tyrosine infusion. Steady-state conditions were

reflected by constancy of the difference between arterial and venous SRA and by low CV (<6%) forarterial SRA (data not presented).

Precise preparation of the [3H]tyrosine infúsate (seeMaterials and Methods) ensured uniform, within-animal infusion rate (mean CV = 2.4%, range = 0.5 to5.2%). Because variation among animals is accounted

TABLE 2

Feed intake, digestibility and nitrogen balance in growing steers fed either 60% (0.6M) or 220% (2.2M)of predicted energy requirement for maintenance1

TreatmentVariableDry

matterIntake,g/dDigestibility,

%Nitrogen

partitioning,g/dIntakeFecalUrinaryRetained0.6M168874.731.19.830.6-9.32.2M600367.9144.247.365.731.2SEM550.71.40.40.41.9Significance

level20.0010.020.0010.0010.0050.005

Variables were measured between d 9 and 16 of each treatment period (four animals per treatment).2Type I error probability.

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PROTEIN TURNOVER IN FED AND UNDERFED STEERS 1081

TABLE 3

Arterial concentration of blood components, metabolites and hormones in growing steers fed either 60% (0.6M)or 220% (2.2M) of predicted energy requirement for maintenance1

TreatmentVariableBlood

componentPackedcell volume,%Hemoglobin,

mg/LBlood

metabolite,mmol/LGlucoseAcetate3-HydroxybutyrateLáclateOxygenPlasma

metabolite,mmol/LNonesterifiedfattyacidsUreaHormone,

nmol/LSeruminsulinSerumIGF-IPlasma

somatotropin0.6M32.91093.090.5670.3590.3526.250.4271.260.04212.70.202.2M28.9963.431.0850.4460.4485.520.1181.760.08424.30.14SEM0.210.060.0270.0130.0100.060.0360.040.0011.20.02Significance

level20.010.020.040.010.040.020.020.030.020.0050.02NS

Values represent average of d 12 and 14 of treatment; nine samples were obtained each day between 1215 and 1615 h (four animals pertreatment). IGF-I = insulin-like growth factor.

2Type I error probability, where NS = nonsignificant at P > 0.10.

TABLE 4

Arterial blood concentration of amino acids in growing steers fed either 60% (0.6M) or 220% (2.2M)of predicted energy requirement for maintenance1

TreatmentAmino

acidTaurineThreonineSerineAsparagineGlutamateGlutamineGlycineAlanineCitrullineValineIsoleucineLeucineTyrosinePhenylalanineHistidineOrnithineLysineArginine0.6M136.6122.890.427.5194.3181.2433.4153.862.6236.790.0152.239.445.1104.3147.7195.089.62.2M\un

olIL119.8136.594.743.9141.3207.5358.5166.687.4286.3131.4178.161.051.8110.7133.8170.9113.2SEM8.56.20.82.06.62.720.51.06.69.34.73.00.80.83.08.84.34.9Significance

level2NSNS0.070.0050.010.03NS0.020.080.060.020.030.0050.03NSNS0.030.05

'Values represent average of d 12 and 14 of treatment; nine samples were obtained each day between 1215 and 1615 h (four animals per

treatment).Type I error probability, where NS = nonsignificant at P > 0.10.

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1082 BOISCLAIR ET AL.

TABLE 5

Hindlimb blood flow, arteriovenous difference and net uptake of respiratory fuels in growing steers fedeither 60% (0.6M) or 220% (2.2M) of predicted energy requirement for maintenance1

TreatmentVariableBlood

flow,L/minArteriovenous

difference,mmol/LGlucoseAcetate3-HydroxybutyrateLactateOxygenNet

uptake,mmol/minGlucoseAcetate3-HydroxybutyrateLactateOxygen0.6M0.5070.1250.3280.139-0.0623.610.0620.1580.068-0.0341.782.2M1.3480.1730.5230.1400.0102.660.2300.7040.1870.0133.59SEM0.0180.0190.0320.0050.0170.270.0160.0110.0040.0100.128Significance

level20.001NS0.05NS0.030.050.020.0010.0050.020.01

'Values represent average of d 12 and 14 of treatment; nine samples were obtained each day between 1215 and 1615 h (four animals per

treatment).2Type I error probability, where NS - nonsignificant at P > 0.10.

for in the statistical model, effects of treatment canbe assessed from the radioactivity and SRA data(Table 7). Arterial levels of [3H]tyrosine, hindlimb AVdifferences for [3H]tyrosine, and arterial and venousSRA were all increased during the period of under-

nutrition. Tracer radioactivity was always higher in

the artery than in the vein (paired t test, P < 0.05,mean of eight observations per infusion), reflectinguptake of tyrosine irrespective of treatment. Similarobservations for the SRA data (P < 0.05) indicate thatrelease of unlabeled tyrosine from the hindlimb occurred concurrently. Clearly, release exceeded uptake

TABLE 6

Blood arteriovenous difference and net uptake of amino acids across the hindlimb in growing steers fedeither 60% (0.6M) or 220% (2.2M) of predicted energy requirement for maintenance1

Arteriovenous difference Net uptake

TreatmentVariableThreonineSerineAsparagine

GlutamateGlutamineGlycine

AlanineValineIsoleucineLeucineTyrosine

PhenylalanineHistidineOrnithineLysine

Arginine0.6M-3.45.10.4

23.1-21.5-7.2

-21.5^3.00.80.3-1.9-2.8

-0.77.0-0.1

-3.02.2M\unol

IL4.811.85.4

23.9-1.26.913.212.510.512.72.2

2.62.96.19.8

7.5SEM130.80.8

1.94.82.8

3.92.31.61.20.4

0.60.41.00.5

1.4Significance

level20.010.0050.01

NS0.030.05

0.010.0050.0050.0010.001

0.0010.03NS0.005

0.03Treatment0.6M-1.72.60.2

11.7-10.1-3.5

-10.4-1.20.40.1-0.9

-1.4-0.43.4-0.2

-1.52.2M\unollmin5.915.97.2

32.4-1.78.8

17.917.014.217.23.0

3.53.88.113.1

10.1SEM1.50.81.0

2.12.23.2

3.62.02.11.40.5

0.50.71.00.8

0.6Significance

level20.020.0010.005

0.020.040.09

0.0050.030.0050.0010.005

0.0010.010.020.001

0.001

'Values represent average of d 12 and 14 of treatment; nine samples were obtained each day between 1215 and 1615 h (four animals per

treatment|.2Type I error probability, where NS - nonsignificant at P > 0.10.

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TABLE 7

Arterial level and hindlimb arteriovenous difference in tyrosine activity and specific radioactivity of tyrosine in arterial andvenous blood in growing steers fed either 60% (0.6M) or 220% (2.2M) of predicted energy requirement for maintenance*

Treatment

Variable 0.6M 2.2M SEM

Significancelevel2

Radioactivity, kBq/LArteryArteriovenous difference

Specific radioactivity,ArteryVein

63.212.8

1.661.28

52.66.0

0.890.82

2.40.5

0.060.05

0.050.02

0.020.03

Variables represent average of d 12 and 14 of treatment; eight samples were obtained each day between 1245 and 1615 h (four animals pertreatment).

2Type I error probability.

during undernutrition because the net AV differencefor tyrosine was negative (Table 6).

Figure 1 illustrates the close agreement betweenestimates of protein kinetics obtained on d 12 and 14of each treatment period and the absence of systematic bias introduced by repeating the measurement of protein turnover. Mean values forhindlimb protein kinetics, averaged for the 2 d ofmeasurement, are summarized in Table 8. Proteinbalance calculated from tyrosine or phenylalanine AVdifferences shifted from net accretion to net proteinmobilization upon the imposition of chronic undernutrition (P < 0.002). Chronic underfeeding reducedhindlimb protein synthesis rates by 49% (P < 0.001)without affecting rates of protein degradation. Undernutrition also resulted in a 48% reduction in thewhole-body turnover rate of tyrosine (P < 0.001). We

estimated that protein synthesis accounts for 13% of

hindlimb energy expenditure, with no indication ofan effect of intake level (Fig. 2).

DISCUSSION

The primary objective of this study was to evaluatethe ability of the AV difference technique to estimatechanges occurring in protein kinetics in the hindlimbof cattle. We chose to vary nutritional status becauseeffects on N balance would be reflected by changes inprotein synthesis and/or degradation. Methodologyalso provided an opportunity to characterize simultaneously hindlimb protein kinetics and nutrientutilization during periods of growth and chronicwasting of skeletal muscle. Three important criteriawere met for the conducting of our study. First,

TABLE 8

Estimates of hindlimb protein kinetics and whole body tyrosine flux in growing steers fed either 60% (0.6M)or 220% (2.2M) of predicted energy requirement for maintenance1

TreatmentVariableHindlimb

protein kinetics,mg/minBalance3TyrosinePhenylalanineSynthesisDegradationWhole-body

tyrosine flux, \unol/min0.6M-6.5-6.735.542.0159.02.2M21.016.769.248.2308.9SEM3.82.54.02.66.3Significance

level20.0020.0010.001NS0.001

Variables represent average of d 12 and 14 of treatment; eight samples were obtained each day between 1245 and 1615 h (four animals pertreatment).

2Type I error probability, where NS = nonsignificant at P > 0.10.3Protein accretion calculation using tyrosine or phenylalanine.

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100

0>

60

40

IO-

-20-20 20 40 60 80 100

DAY 12 PROTEIN KINETICSmg/min

FIGURE 1 Comparison between estimates of rates ofprotein synthesis (circles), protein balance ¡squares) andprotein degradation (triangles) obtained on d 12 and 14 ofeach period. Treatments were either 60% (open symbols) or220% (closed symbols) of the energy requirement for maintenance. Line of equality is depicted for comparison.

steady-state conditions of nutrient supply were approximated by feeding at hourly intervals. Thus, arterial levels and hindlimb utilization of variousnutrients were constant over time (data notpresented). Second, measurements were made over asufficient time interval to allow extrapolation to theentire day. Finally, animals were adapted to the levelsof intake prior to measurements. This is particularlyimportant when severe metabolic challenges such asfood deprivation or chronic undernutrition are imposed. Daily N losses during the last week of underfeeding were essentially constant (data not presented),thus confirming steady-state conditions with regardto N metabolism.

Respiratory fuel metabolism. Allowing for differences in feed intake and body weight, present valuesfor external iliac blood flow and hindlimb net exchanges of oxygen, glucose, lactate, acetate and3-hydroxybutyrate in well-fed steers (Table 5) werecomparable to those in previous reports for youngcattle and sheep (reviewed by Pethick et al. 1984).Assuming complete oxidation, glucose (corrected forlactate), acetate and 3-hydroxybutyrate accounted for41, 39 and 24% of hindlimb oxygen consumption,respectively, and agreed with derived quotients of 41,32 and 21% for data on metabolism of the hindhalf(both hindquarters) of beef steers fed at a similar level(Eisemann et al. 1988).

Chronic undernutrition produced major changes inhindlimb metabolism. First, the reductions in blood

HINDLIMB ENERGY EXPENDITURE

mmol ATP/min

FIGURE 2 Relationship between energy cost of proteinsynthesis and energy expenditure in the hindlimb in steers.The ATP equivalents for protein synthesis and energy expenditure (based on oxygen consumption) were calculatedaccording to the stoichiometric relationships outlined inMaterials and Methods. Treatments were intakes of either60% (open symbols) or 220% (closed symbols) of the energyrequirement for maintenance. Best fit equation is y = 0.34 ±0.13X, (r2 = 0.84, P < 0.0001).

flow and in net nutrient utilization (Table 5) wereundoubtedly related to a considerable decline in anabolic processes. Animals reached a new metabolicequilibrium characterized by increased reliance onendogenous substrates, as demonstrated by theoxygen quotients across the hindlimb. Thus, glucose(corrected for lactate) and acetate accounted for lessthan 16 and 18%, respectively, of the hindlimboxygen consumption during underfeeding. The bulkof this deficit was probably made up by increasedNEFA utilization, consistent with previous observations of major increases in mobilization and oxidativeuse of NEFA in the whole body and hindlimb ofunderfed ruminants (Bell and Thompson 1979,Pethick et al. 1983). As previously shown in sheep(Pethick et al. 1983), the hindlimb became activelyinvolved in the conservation of glucose carbon duringundernutrition by recycling -25% of net uptake

through lactate return (Table 4).Somatotropin is a major positive determinant of

circulating levels of IGF-I in well-fed animals

(McGuire et al. 1992), yet undernutrition caused a50% reduction in the circulating levels of IGF-I de

spite the trend for an increase in plasma levels ofsomatotropin (Table 3). Although repeatedly observedin cattle, the mechanism underlying this uncouplinghas not been completely defined in ruminants. Energy

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PROTEIN TURNOVER IN FED AND UNDERFED STEERS 1085

and protein supply have been implicated, but additional factors such as the low insulin levels mightalso play a role (McGuire et al. 1992).

Hittdlimb nitrogen metabolism. Although ruminant muscle possesses the ability to metabolize anumber of amino acids (Bergman and Pell 1984),protein accretion remains the major determinant ofamino acid balance. Hence, our growing animals exhibited positive AV differences for most amino acidsacross the hindlimb (Table 6). The direction and magnitude of the AV differences broadly agree with datareported for younger, slower-growing steers (Bell et al.1975). As expected, they are larger than thosereported for animals with static protein stores such asolder cattle (Huntington and Prior 1985) and maturesheep fed at maintenance level (Bergman and Pell1984, Teleni et al. 1986). With underfeeding, mobilization of amino acids from endogenous pools such asmuscle protein occurred across the hindlimb (Table6). It is particularly notable that the outputs (negativeAV differences) of glutamine, glycine and alaninewere the greatest, given the ability of these aminoacids to support hepatic gluconeogenesis (Bergmanand Pell 1984).

Net exchanges of the aromatic amino acids can beused to calculate hindlimb N retention, which canthen be related to whole-animal N retention. Valuesfor tyrosine and phenylalanine uptake suggest thatthe hindlimb contributed 16 and 12% of whole-bodyN retention, respectively, at the 2.2M intake, and 16and 17% of whole-body N loss, respectively, at the0.6M level. Because hindlimb muscles represent-15% of whole-body skeletal musculature, these estimates further add to our confidence in the quantitative validity of measurements of tyrosine andphenylalanine uptake. As subsequently discussed,this is critically important for reliable estimation ofhindlimb protein degradation.

Effects of treatment on protein kinetics. Chronicunderfeeding was clearly associated with a reductionof protein synthesis in the hindlimb (Table 8). Similarobservations have been reported by others for ruminant muscle under various conditions of nutritional deprivation, using either the AV difference(Harris et al. 1992, Oddy et al. 1987, Pell et al. 1986)or the label incorporation technique (Patureau-Mirandet al. 1985). Depression of rates of muscle proteinsynthesis seems to be a rather general consequence ofundernutrition because it has also been documentedin humans (McNurlan and Garlick 1989) and rodents(Millward et al. 1976).

The primary factor(s) causing the reduction ofhindlimb protein synthesis in underfed steers remainsto be elucidated. Most mechanistic studies must beconsidered cautiously because measurements ofprotein kinetics occurred before metabolic adaptations to food deprivation or undernutrition were completed. Nevertheless, the depression in circulating

levels of insulin could be involved, as shown in youngrats in the postabsorptive state (reviewed inMcNurlan and Garlick 1989). However, insulin hadno effect on rates of protein synthesis in humansfasting for 12 to 60 h (Fryburg et al. 1990, Gelfand andBarrett 1987) or young lambs deprived of food for 24to 48 h (Douglas et al. 1991, Oddy et al. 1987), suggesting that insulin plays a lesser role in the regulation of muscle protein synthesis in these species.

In contrast to results with insulin, short-term infusions of IGF-I elevated rates of protein synthesis inskeletal muscles, heart and liver of food-deprived

lambs (Douglas et al. 1991). In vitro studies alsosuggest that physiological levels of IGF-I, but not

insulin, stimulate protein synthesis in myotubes derived from satellite cells (Roe et al. 1989) and musclecell lines (Gulve and Dice 1989). Circulating levels ofIGF-I are not acutely modulated by nutrient supplyover the course of a day, as is insulin concentration.Therefore, the fall of IGF-I levels during chronic un

dernutrition (Table 3) is consistent with a role in thelong-term modulation of protein metabolism(McGuire et al. 1992).

Underfeeding had no effect on rates of proteindegradation (Table 8). Other studies using the AVdifference technique in ruminants have reported thatunderfeeding or starvation resulted in increased (Oddyet al. 1987, Teleni et al. 1986), decreased (Harris et al.1992) or unaltered (Pell et al. 1986) rates of proteindegradation. Apart from the difficulties associatedwith the derivation of protein degradation (see below),this inconsistency could simply reflect the variety ofexperimental conditions used (e.g., age of animals,severity and duration of undernutrition). For instance,Oddy et al. (1987) deprived young, milk-fed lambs offood for 24 to 48 h, whereas Pell et al. (1986) deprivedmature sheep of food for 3 d, before assessing proteinkinetics. Young lambs likely experienced a moreserious metabolic challenge than mature sheep at thetime of measurement. This interpretation is supported by observations in rats showing that muscleprotein degradation rates do not increase until a relatively severe metabolic situation develops (Millwardet al. 1976) and that the time before accelerated pro-

teolysis is inversely related to the degree of leanness(Goodman et al. 1984).

Protein kinetics method: methodological considerations. The present method relies on precise quantification of the fluxes of mass and radioactivity of aselected amino acid across the hindlimb. Any errorsin the primary determinants (blood flow and AVdifferences) are multiplicative and have considerableimpact on the estimates of protein kinetics. Hence,scrupulous analytical procedures and repeated sampling are needed to obtain accurate estimates. Theseconsiderations are particularly critical for amino acidsdisplaying small AV differences, such as tyrosine andphenylalanine (Table 6).

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The amino acid chosen to monitor proteinturnover determines the complexity of the derivations and the precision of the estimates. Ideally, itsonly metabolic fate would be incorporation intoprotein. This condition is not essential as long asintermediary metabolites exchanging with muscle areknown and can be reliably quantified. Indeed, leucinehas been a popular choice to study protein turnoverbecause of its large AV difference (Table 5). However,use of this amino acid requires multiple isotope infusions and quantification of the fluxes and concentrations of all possible products of catabolism (a-ketoisocaproate and carbon dioxide) across thehindlimb (Oddy and Lindsay 1986, Pell et al. 1986).Analytical errors associated with these additionalmeasurements would tend to offset any apparent gainin accuracy from the larger AV differences. We chosetyrosine because the aromatic amino acids are notcatabolized in muscle (Chang and Goldberg 1978). Inretrospect, phenylalanine would have been a slightlybetter choice because it has a 1.4-fold greater abundance in carcass protein than does tyrosine (Williams1978). This is because, for a non-catabolized amino

acid, the magnitude of the AV differences in mass andradioactivity at a given blood flow, and hence theability and accuracy of this method to detecttreatment differences, are functions of its content inthe muscle.

Unlike traditional isotope incorporation methods(Reeds 1992), our approach permits repeated measurements on the same animal. Figure 1 illustrates thevariation in the estimates of protein synthesis,balance and degradation obtained on two differentdays. Any deviation from the line of equalityrepresents the cumulative errors incurred in the estimation of blood flow, tyrosine radioactivity andtyrosine concentration. The precision of the variousestimates is very acceptable, particularly when considering all potential sources of error. Rates of proteindegradation are more variable, being obtained bydifference between protein synthesis and balance.Isotope recycling and consequent underestimation ofprotein synthesis have been documented previously(reviewed by Reeds 1992). The relative constancy ofarterial SRA and AV difference for [3H]tyrosine (data

not shown) suggests that recycling was negligible overthe course of our infusions. Significant recycling inthe hindlimb would also lead to lower estimates ofprotein synthesis on the second day. The lack of anysystematic error (Fig. 1) and the absence of a dayeffect in the initial statistical analysis again indicatethe minimal impact of this phenomenon.

Accurate estimation of protein synthesis has beenprecluded by the inability to measure the specificradioactivity of the precursor pool providing theamino acids for protein synthesis (Reeds 1992). Thisproblem is common to all methods, although tovarying degrees. Determination of the aminoacyl-

tRNA or ribosome nascent peptide specific radioactivity would provide the most nearly correct estimate.However, the complexity of the chemical analysesand the biopsy of muscle and associated stress makethis option incompatible with in vivo experimentation of the present type. An alternative is to samplea distal pool that might correctly reflect the precursorpool specific radioactivity. Previous investigationswith [3H]tyrosine are particularly disconcerting in

this regard. Incorporation of label into tissue proteinwas consistent with tRNA charging occurring from apool of tyrosine in rapid equilibrium with the freeintracellular pool (rat diaphragm; Li et al. 1973), withthe extracellular pool (rabbit muscle; Lobley et al.1980b) or with a mixture of the two (myocardium;O'Hara et al. 1981).

In the absence of conclusive evidence to the contrary, we consider the intracellular amino acid pool torepresent the true precursor pool. We are not aware ofstudies in which the specific radioactivity of freetyrosine was measured simultaneously in arterial andvenous blood serving a muscle bed and in muscleitself (intracellular pool). Nevertheless, specific radioactivity of free tyrosine was always higher in venous(jugular or ear vein) blood than in the musclehomogenates when infusing the tracer in the con-tralateral vein (A-V mode) (Lobley et al. 1980a and1980b). Therefore, the venous specific radioactivitywas chosen to derive rates of protein synthesis because it should reflect more accurately the intracellular specific radioactivity. Systematic errors incurredby using the wrong specific radioactivity are tolerablefor protein synthesis, assuming that the relation between the sampled pool and true precursor pool is notaltered by treatment. However, systematic errors inprotein synthesis can either mask or create treatmenteffects on estimates of protein degradation when calculated by difference between protein balance andsynthesis. Therefore, we would expect this techniqueto be somewhat less powerful in detecting treatmentdifferences when protein degradation is the main affected variable. In the present study, effects oftreatment on protein synthesis and degradation wereessentially the same irrespective of the specific radioactivity used (arterial, venous or an average of thetwo). Obviously, the numerical discrepancy amongthe estimates of synthesis and degradation producedby using these various specific radioactivities wasexaggerated during the underfeeding period, when thedifference between the arterial and venous specificradioactivity of tyrosine was much greater (Table 7).

Two additional observations support the validity ofthe AV difference technique. First, we observed astrong association between protein synthesis andenergy expenditure in the hindlimb. In the wholeanimal, protein synthesis consistently represented-15% of energy expenditure, across a wide range of

intakes, in pigs (Reeds et al. 1985), sheep (Harris et al.

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1992) and cattle (Lobley et al. 1980a and 1987).Second, we can compare our estimate with valuesobtained in growing cattle with the isotope incorporation method. Using either plasma or intracellularSRA, fractional synthetic rates of protein reported forskeletal muscle of fed animals (control group only)ranged from 0.6 to 2.2 %/d with leucine (Eisemann etal. 1989), 1.6 to 2.1 %/d with [3H]tyrosine (Lobley etal. 1980a) and 0.6 to 2.8 %/d with [3H]phenylalanine

(Early et al. 1990). By converting the daily rate ofprotein synthesis (Table 8) into a fraction of theprotein mass and assuming muscle consumes thebulk of [3H]tyrosine, we obtain a fractional synthetic

rate of 3.6 %/d at the high level of intake. Thisestimate might, however, be significantly discountedin view of the high rates of protein synthesis reportedfor bone and skin (Lobley et al. 1980a). Thus, the AVdifference method suffers from a certain degree ofnonspecificity, depending upon which venous site issampled. Quantifying protein kinetics across the deepfemoral vein preparation would eliminate some ofthis limitation, because the deep femoral vein drainsmuscle almost exclusively (Teleni and Annison 1986).

In conclusion, underfed animals exhibited reducedhindlimb blood flow and decreased utilization ofrespiratory fuels and amino acids. We demonstratedthat the hindlimb loss of N during chronic under-nutrition could be entirely ascribed to a reduction inrates of protein synthesis. This study also attested tothe validity and applicability of the AV differencetechnique to estimate protein turnover in largeanimals. Specifically, estimated rates of protein synthesis were related to oxygen consumption, agreedwith published estimates and changed as predictedwith nutritional status. Hindlimb tyrosine balancewas qualitatively and quantitatively related to whole-animal N balance. Both parameters were estimatedwith adequate precision. This method confers theunique advantage of simultaneous estimation ofprotein degradation. Equally important are thepotential gains in statistical power achieved byrepeating the measurements within and across treatments on the same animal. Finally, because bloodflow and concentration of tracer and tracéeaminoacids are the primary variables, any advances in theirmonitoring such as continuous blood flow estimationand integrated blood sampling would further improvethe precision and reliability of the AV technique.

ACKNOWLEDGMENTS

We wish to thank M. A. McGuire, R., Slepetis, W.English, D. Ross, D. Dwyer and K. Johnston for assistance in data collection and chemical analyses andD. Ceurter for preparation of the manuscript. We arealso grateful to the National Institute of Diabetes,Digestive and Kidney Diseases for donating the antibodies used in somatotropin and IGF-I assays.

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