effect of dietary branched-chain /-reto acids on hepatic branched

Effect of Dietary Branched-chain /-Reto Acidson Hepatic Branched-chain /-Keto AcidDehydrogenase in the Rat1

BALWANT S. KHATRA,- RAJENDER K. CHAWLA,3ALLAN D. WADSWORTH ANDDANIEL RUDMANDepartments of Surgery and Medicine, Emory UniversitySchool of Medicine, Atlanta, Georgia 30322

ABSTRACT Male albino rats (80-100 g) were tube-fed for 3 days with(a) a complete purified amino acid diet minus valine, or this diet containing 70 to 210 /Â¿mole/gof valine (1 to 3 times the minimal daily requirement, MDR) or equimolar amounts of its a-keto analogue (KIV); (b) complete diet minus leucine, or this diet containing 85 to 225 /^mole/g of eitherleucine ( 1 to 3 times the MDR ) or its a-keto analogue ( KIC ); (c ) complete diet minus valine, leucine and isoleucine, or this diet containing 63to 170 >xmole/g of these amino acids (1 to 2 times the MDR) or theira-keto analogues (KIV, KIC, KMV). Liver and kidney were then assayedfor dehydrogenase activity towards these substrates: KIV, KIC, KMV,pyruvate, and a-ketoglutarate. Both the branched-chain amino acids (BCAA)and their a-keto analogues (BCKA) stimulated the activity of branched-chain hepatic dehydrogenases. BCKA were 2 to 9 times more potent thanBCAA in this respect. The effect was specific for enzyme and organ, sincedehydrogenase activity for a-ketoglutarate and pyruvate in liver, and dehydrogenase activity for BCKA, pyruvate and a-ketoglutarate in kidney,were not increased. Dietary BCKA (1 to 2 times MDR) accelerated thedecarboxylation of KIC by slices of liver 2 to 6 times without altering therate of transamination to leucine. Decarboxylation of KIC by kidney andmuscle slices was unaffected. The stimulation of hepatic branched-chaindehydrogenase by BCKA may play a role in the limited nutritional efficiency of these nitrogen-free substitutes for the BCAA compared to a-ketoanalogues of other essential amino acids. J. Nutr. 107: 1528-1536, 1977.INDEXING KEY WORDS Branched-chain keto acid dehydrogenasesâ€¢branched-chain keto acids â€¢branched-chain amino acids

Branched-chain keto acids [a-ketoiso- Rational clinical use of branched chainvaleric acid (KIV), a-ketoisocaproic acid keto acids (BCKA) will require informa-(KIC), and a-keto-/3-methylvaleric acid tion about the efficiency of each BCKA as(KMV)] can substitute for the correspond- a substitute for its corresponding branched-mg essential amino acids, valine, leucine chain amino add (BCAA). Percent effi-and isoleucine respectively, in the immature rat to promote growth (1-3), and inadult man to maintain nitrogen balance Receivedfor publicationNovember3, me.(3, 4 ). Recently these substances have ' supported by USPHS grants AMISTSSandbeen proposed as dietary supplements tO K^0p>r0e^ntaddress: Department oÃ Physiology,protein-deficient diets for patients with rs,ch001of ÃÃA1"6'Vanderbllt university, Nashville,

l i_ â€¢ rr* â€¢ i ^ r* \ â€¢*^nn<?sst?6 o7*.OÃÂ¿.renal Or hepatic insufficiency (5-7). Â»Address all correspondence to this author.

1528

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BRANCHED-CHAIN Â«-KETOACID DEHYDROGENASE 1529

ciency of substitution (2 ) has been definedas:

Moles of amino acid requiredto produce a specifiedphysiologic response (e.g.,

growth rate or X-balance)Moles of keto acid required to

produce the same response

X 100

In the growing rat, efficiency of K1V as asubstitute for valine varies from 80% foran intake corresponding in moles to 10%the minimal daily requirement ( MDR ) 4of valine to 307Â»for higher intakes (2).Only when rats are fed a valine-free dietcontaining 210 ^mole KlV/g, equivalentin moles to 3 times the MDR of valine, ismaximal growth achieved. The efficiencyof KIC is even poorer, being 25% to 30%at levels of intake corresponding to 10%to 200^0 the MDR of leucine (9). Theselow efficiency values for KIC and KIVcontrast to the substantially higher valuesfor a-keto derivatives of methionine andphenylalanine (10, 11).

Only two metabolic pathways are available to the BCKA: transamination andoxidative decarboxylation. In the fed rat,the enzyme responsible for the formerprocess occurs largely in muscle and kidney, and that responsible for the lattermainly in liver ( 12). Efficiency of thea-keto acid as a substitute for the corresponding BCAA will depend on the ratio:velocity of transamination/velocity of decarboxylation. Thus any factors which retard transamination or accelerate decarboxylation will reduce efficiency, and viceversa. Wohlhueter and Harper ( 12 ) haveshown that addition of BCAA to the dietof rats at a level 7 to 9 times the MDRincreases the specific activity of the hepaticbranched-chain dehydrogenase which catalyzes the oxidative decarboxylation ofBCKA. Since BCKA are the metabolicproducts of the BCAA and are the substrates of the dehydrogenase, it is possiblethat some or all of the stimulation of thedehydrogenase by BCAA described bythese workers was actually caused bytheir a-keto acid derivatives. If so, thenaddition of BCKA to the diet of rats willhave an even greater effect in stimulatinghepatic branched-chain dehydrogenase

than will the corresponding BCAA. Sucha postulated property of the BCKA couldexplain their low efficiency as substitutesfor BCAA.

The primary objective of the presentstudy was to compare how dietary BCKAand their corresponding amino acids influence the specific activity of the branched-chain dehydrogenase in rat liver. For completeness, branched-chain dehydrogenasewas also measured in kidney, and twoother types of dehydrogenase activity (forpyruvate and a-ketoglutarate ) were assayed in both liver and kidney. In addition,experiments were done to learn how dietary BCKA influence the decarboxylationand transamination of KIV by slices ofliver, kidney and muscle.

MATERIALS AND METHODS

The amino acid composition of eachbatch of diet3 was confirmed by analysisof a sample of diet on an amino acidanalyzer.Â«KIV, KIC, and KMV were synthesized according to the procedure ofWeygand, Steglich, and Tanner (13). Thehomogeneity and identity of each compound were established by elemental analysis, and by thin-layer Chromatographieand gas Chromatographie analyses (2).

Male, albino, Sprague-Dawley rats, 4weeks old and 70 to 80 g in weight, werehoused in individual raised wire-mesh bottom cages and were offered water adlibitum.

Four types of experiments were performed to measure the effect of dietaryBCAA and BCKA on branched-chain dehydrogenase activity in tissue homogenates.Each experiment utilized 4 to 7 groups ofrats; there were 6 to 8 rats per group.Every experiment was repeated 2 to 3times. The experimental design is explained by tables 1 and 2. In each experiment, the rats ate ad libitum completepurified amino acid diet for an initial 3

Â«Minimum Daily Requirement (MDR) is defined asthe smallest concentration of the specified nutrientin an otherwise complete diet which supports maxl-mal growth rate (8).Â»Purified amino acid diet (8) omitting L-valine,i.-leucine. and L-isoleucine, and these amino acidswere purchased from ICN Nutritional BiochemicalsCo., Cleveland, Ohio. Rats were from ARS SpragueDawley, Madison, Wisconsin. Trifluoroacetic acid anhydride for synthesis of Â«-keto acids was from Eastman Kodak Co., Rochester, X.Y. All solvents andrenitentÂ« were of analytical grade.

Â»Beckman 120 C Amino Acid Analyzer. BeckmanInstrument Co., Anaheim, Calif.


1530 KHATRA, CHAWLA, WADSWORTH AND RUDMAN

TABLE 1Outline of four of the five experiments

Substrate

Exp.no.1

234

5Dietary

variablesundercomparisonValine

vs KIVLeucine vs KICBCAA vs BCKABCAA vs BCKABCAA vs BCKAOrgan

studied1a-ketoLiver

Kidney Muscle KICZ KIV3 KMV4 vatetarate+

+

1Tissue homogenates were used in experiments 1 to 4 and tissue slices in experiment 5. 2a-ketoiso-caproic acid (KIC). *a-ketoisovaleric acid (KIV). *a-keto-/3-methylvaleric acid (KMV).

days of acclimitization. During the controlperiod (5 days) and experimental period(3 days), food was given through a stomach tube in the form of a constantlystirred slurry (70 g food made up to 100ml with water). Total amount of food givenper day was 8% to 9% of average todyweight of rats of the same age eating thecomplete diet ad libitum, and was administered in three portions at 08:00, 14:00and 20:00 hours. This represents the average daily food intake of normal growingmale albino Sprague-Dawley rats fed adlibitum. Rats were weighed daily for 8days and were killed between 08:00 and09:00 hours on day 9 by cervical dislocation. Liver and kidneys were removed andassayed for branched-chain dehydrogenaseactivity according to the procedure ofWohlhueter and Harper (12) with slightmodifications. The reaction was carried outin a total volume of 300 julof medium containing NAD, Coenzyme A, potassiumphosphate buffer, pH 6.8, MgCl2, EDTA,Na2CO3, and keto acid in the concentrations specified (11) and in addition: 1 to2 X IO5 dpm of [l-14C]keto acid for assays of liver and 1 to 2 X IO6dpm for thoseof kidney and muscle; 50 Â¿Jof 0.25 Msucrose nomogenate containing 10 mg tissue (wet weight ) ; 4,000 units catalase; and1 /Â¿mole thiamin pyrophosphate. Themedium was incubated at 37Â°for 20 minutes. Reaction was then terminated, andquantity of 14COÂ¿was measured (12).Under the conditions of the assay, linearrelationships were observed oetweenamount of 14COj formed and quantity oftissue homogenate added, and between

amount of 14CO2produced and duration ofincubation. One unit of enzyme activitywas defined as formation of one /Â¿moleof14COÂ¿in 1 hour at 37Â°under the experimental conditions and "specific activity" as

units of activity per g of tissue (wetweight).

Experiment 5 examined the effect ofBCAA- and BCKA-containing diets on thedecarboxylation and transamination of[1-14C]KIC by slices of liver, kidney andmuscle. Dietary conditions are given intables 1 and 2. Between 08:00 and 09:00hours on day 9, the rats were killed. Sections of liver, kidney and psoas musclewere sliced with a Mcllwain tissue chopper set at 0.3 mm. Ten to forty milligramsof sliced tissue was suspended in 0.5 mlfresh rat plasma containing 0.25 to 2.5mM [1-14C]KIC (1 to 5 x 10' dpm/>mole).The incubation was carried out in a Teflontest tube fitted snugly into a scintillationvial containing 0.5 ml of l M methylbenze-thonium hydroxide.7 The Teflon tube wasperforated horizontally at a height suchthat the perforations did not communicatewith the interior of the scintillation vialuntil after the incubation was completed,when the tube was lowered into the vial.After addition of sliced tissue to plasma,the tube was gassed with 95% O2-5%COj, capped and incubated with constantshaking for a specified time. Two-tenthsmilliliter l N PLSO, was then injectedthrough the cap and the tube lowered intothe vial. After 1 hour of further incubationto collect all 14COL.in the base, the tubes

~Xi'\v England Nuclear. Boston, .Muss.


BRANCHED-CHAIN a-KETO ACID DEHYDROGENASE 1531

were removed. Scintillation fluid wasadded to the vial and 14CO2measured bya liquid scintillation counter. Content ofthe incubation tube was hydrolyzed for 18hours in 6 N HC1 at 110Â°in vacuo and

dried. The hydrolysate was then appliedto a 0.5 X 3 cm Dowex column preparedby packing a Pasteur pipette with Dowex50 WX 8-200, H+ form, supported withglass wool; the column was efuted with 1N HC1. [1-14C]KIC emerged in the first 3ml of effluent and [l-14C]leucine eluted between 4 and 12 ml of cumulative volume.The latter fraction was collected and a 3ml aliquot, mixed with 10 ml of Bray's 7

solution, was counted at 60% efficiency.The statistical analyses of experimentaldata were performed using Students' t test

(14).

RESULTS

Experiment 1 (table 3). During the control period, rats were tube-fed completediet containing 70 /Â¿molevaline/g andgained weight at an average of 5.1 g/day.Removal of valine during the experimentalperiod caused weight loss of 2.3 g/day.The growth failure was fully corrected byaddition of 140 to 210 BinÃ³levaline/g tothe valine-free diet and partially correctedby 140 to 210 /Â¿moleKlV/g. At the end ofthe experimental period, hepatic branched-chain dehydrogenase activity was measured with KIV as substrate. Specific andtotal hepatic activity was not increasedsignificantly by addition to the valine-freediet of 70, 140 or 210 /tmole/g of valine(1, 2 or 3 times the MDR). In contrast,rats eating a diet containing 140 or 210/xmole KlV/g showed a 50% to 60% increase in specific and total hepaticbranched-chain dehydrogenase activitycompared to rats eating a valine-free diet.

Experiment 2 (table 4). Relationshipssimilar to those in experiment 1 werefound when leucine was replaced by KIC,hepatic branched-chain dehydrogenase activity now being measured with KIC assubstrate. Specific and total hepatic activities were similar in rats tube-fed leucine-free diet, or this diet containing 170 /Â¿moleleucine/g (2 times the MDR). Only whenleucine was fed at 255 /Â¿mole/gdiet (3times the MDR), did hepatic branched-chain dehydrogenase specific and total

TABLE 2Protocolfor the 6 experiments1

Exp.no. Group Experimental period (3 days)

1 1 Val free synthetic amino acid diet2 Val free synthetic amino acid diet

+ 70 Â«molesVal/g diet3 Val free synthetic amino acid diet

+ 70 AmÃ³lesKlV/g diet4 Val free synthetic amino acid diet


+ 140 Â«molesKlV/g diet6 Val free synthetic amino acid diet


+ 210 Â«molesKlV/g diet2 1 Leu free synthetic amino acid diet

2 Leu free synthetic amino acid diet+ 85 Â«molesLeu/g diet

3 Leu free synthetic amino acid diet+ 85 Â«molesKIC/g diet





3 1 Leu, Val, Ile free synthetic amino acid diet2 Leu, Val, Ile free synthetic amino acid diet

+ 85, 70 and 63 Â«molesLeu, Val and Herespectively per g diet

3 Leu, Val, Ile free synthetic amino acid diet+ 85, 70 and 63 Â«molesKIC, KIV andKMV respectively per g diet

4 Leu, Val, Ile free synthetic amino acid diet+ 170, 140 and 126 Â«molesLeu, Val andlie respectively per g diet

5 Leu, Val, He free synthetic amino acid diet+ 170, 140 and 126 Â«molesKIC, KIVand KMV respectively per g diet

4 1 Complete synthetic amino acid diet2 Leu, Val, Ile free synthetic amino acid diet3 Leu, Val, Ile free synthetic amino acid diet

+ 170, 140 and 126 Â«molesLeu, Val andlie respectively per g diet

4 Leu, Val, He free synthetic amino acid diet+ 170, 140, 126 Â«molesKIC, KIV, and

KMV respectively per g diet.5 Same experimental design as exp. 3

1A 5-day control period during which the complete purifiedamino acid diet was fed preceeded the experimental period ineach experiment.

activity increase to a significant degree(307c, P < 0.01) over that in rats fedleucine-free diet. In contrast, addition of170 jumÃ³leKIC/g to the leucine-free dietcaused a 30% increase in hepatic activity,both specific and total although the increase was not significant with diets containing 255 /Â¿moleKIC/g; moreover, he-



TABLE 3Influence of valine and a-ketoisovaleric acid (KIV) on branched-chain dehydrogenase activity

in rat liver (experiment 1)

Groupno.1234567Dietary

contentVal

KIVliinoks/g0

07000

7014000

14021000

210Body

wt9127

Â±1.63"132ÃŒ1.30"12.5il.21Â»141

i1.85e133il.20"14Ãœ2.02'141

Â±1.99eLiver

wtgÃ“.84ÃŒ0.18116.23Â±0.07"0.06ÃŒ0.226.'5.75ÃŒ0.106o.26Â±0.09Â°o.SOiO.lo65.40i0.12Â°KICdehydrogenaseactivityUnits/total

liver/kgbodywtI,148.03il40.57Â°'c707.84Â±

85.87Â°1,314.63Â±159.16C984.33

i82.78'1,677.91 i9.5.3l11l,211.60i

44.54el,700.56i72.38"

Values represent meaniSE (n = 6). In each column, means not having a common superscript letter aresignificantly different (P < 0.05).

patic specific and total dehydrogenase increase in the hepatic enzyme activitiesactivities were 60% greater than in rats than did diets containing the equimolareating leucine-free diet. Hepatic enzyme amounts of BCAA in most cases. The stim-activities were significantly greater in rats ulation of hepatic dehydrogenase was 1.2fed KIC than in those receiving molar to 2.3 times larger for the three BCKAequivalent amounts of leucine at 85 and than for the three BCAA at each level of170 /Â¿moles/gbut not at 255 /Â¿moles/g. intake.

Experiment 3 (table 5). Hepatic dehy- Experiment 4 (fig. 1). In this experiment,drogenase was assayed with both KIC and dehydrogenase activities were measuredKIV as substrates. Rats receiving diets in both liver and kidney simultaneouslycontaining the mixture of three BCAA at with five different keto acids as substrate,1 and 2 times the MDR showed, respec- namely KIC, KIV, KMV, pyruvate andtively 30% and 100% increases in specific a-keto glutarate. As in experiment 3, weand total hepatic dehydrogenase activity, studied the effect of adding to the BCAA-as compared to the activity observed in free diet either all three amino acids, orBCAA-free group. The increase was sig- all three keto acids, at levels correspond-nificant in most cases. Diets containing the ing to 2 times the MDR. In liver (fig. 1A),three BCKA caused a significantly greater the specific and total dehydrogenase ac-

TABLE 4Influence of leucine and a ketoisocaproic acid (KIC) on branched-chain dehydrogenase activity

in the rat liver (experiment a)

Groupno.1234567Dietary

contentLeu

KICnmoles/g0

08500

8517000

17025500

255Body

wt0132.86Â±2.14Â»139.64iO.696148.93Â±2.12C150.50i2.04c151.79i3.67'.Â»154.57

Â±1.77''151.60iO.70'.Â»Liver

wtg4.92i0.18Â°5.87ÃŒ0.17"5.58ÃŒ0.1566.39i0.19c6.79i0.15'.<<6.58i0.10Â£.Â°6.92ÃŒ0.17Â»KICdehydrogenaseactivityU

nits / total liver/kgbodywt919.91

i94.56Â°'e721.97ÃŒ47.98Â°898.80ÃŒ

52.39"l,038.84i36.22'.Â»I,178.18il20.79Â°.Â«l,248.68i

68.37Â«.'1,562.19Ãœ38.41'

Values represent meaniSE (n = 7). In each column means not having common superscript letters aresignificantly different (P < 0.05).



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KIC KIV KMV PyruvOW Â«x-KG

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Fig. l Effect of simultaneous replacement ofall dietary branched chain amino acids bybranched chain keto acids (2 times the MDR)on dehydrogenase activity for a-ketoisocaproicacid, (KIC), a-ketoisovaleric acid (KIV). o-keto-/3-methyvaleric acid (KMV), pyruvate and a-keto-glutarate in rat liver (A) and rat kidney (B).Each bar expresses the a-keto acid dehydrogenaseactivity in livers/kidneys of rats fed experimentaldiet compared to that of rats fed complete purified amino acid diet (taken as 100). Averagevalues for six rats in each group are shown; linesindicate SEM. Details of experimental diets arepresented in table 2 (exp. 4); in each case openbars, slant bars, dotted bars and dark bars represent experimental diet No. 1, 2, 3 and 4 respectively.

tivities for KIC, KIV and KMV rose about20% in response to 2 times the MDR ofthe BCAA and about 130% in response tomolar equivalent amounts of the BCKA.Hepatic dehydrogenase activity for pyruvate and a-ketoglutarate did not changesignificantly. In kidney (fig. IB), the addition of BCAA had no effect on activitytowards any of the five keto acids. Feeding of the three BCKA, however, led to a40% reduction in total and specific renaldehydrogenase activities for KIC, KIV andKMV; activity for pyruvate was unaffected,and the activity for a-ketoglutarate wasdepressed.



TABLE 6Effect of dietary branched chain keto acids on metabolism of [./-"C] a-keioisocaproic acid by slices

of rat liver, kidney and muscle (experiment 5)

Diet'BCAA-freeBCAA-free

+ 1 MDRBCAABCAA-free

+ 2 MDRBCAABCAA-free

+ 1 MDRBCKABCAA-free

+ 2 MDR BCKACone

KIC0.23

mu2.0mÂ«j0.25

IBM2.0mx0.25

mu2.0mil0.25

mu2.0mil0.25

mil2.0 milLiverCO>1.2

Â±0.22.0Â±0.41.3

Â±0.12.2Â±0.41.5

Â±0.32.6Â±0.22.6

Â±0.4Â«5.3Â±0.6*5.5

Â±0.9Â»8.1 Â±1.3*Leucine0.6

Â±0.091.2Â±0.30.7

Â±0.11.4Â±0.20.8

Â±0.11.5Â±0.70.7

Â±0.071.0Â±0.30.5

Â±0.081.1Â±0.1KidneyCO,0.6

Â±0.091.2Â±0.10.4

Â±0.051.7Â±0.20.8

Â±0.11.4Â±0.20.5

Â±0.091.3Â±0.20.5

Â±0.11.3 Â±0.2Leucine2.7

Â±0.34.3Â±0.51.9

Â±0.25.0Â±0.82.9

Â±0.33.5Â±0.53.6

Â±0.44.7Â±0.62.2

Â±0.24.8 Â±0.5MuscleCOÂ«0.07

Â±.0060.14Â±.0070.06

Â±.0050.15Â±.0090.06

Â±.0060.12Â±.010.05

Â±.0070.16Â±.0090.08

Â±.0050.13Â±.009Leucine2.2

Â±0.54.6Â±0.41.9

Â±0.25.0Â±0.93.0

Â±0.34.9Â±0.62.5

Â±0.45.2Â±0.71.9

Â±0.33.7 Â±0.4

Branched chain amino acids (BCAA), branched chain keto acids (BCKA) ; a-keto isocaproic acid (KIC). Dietary conditionsare given in table 3. Values represent pmole 14Crecovered as "CO: or as "C leucine per g tissue (wet wt) per hour (Â»vgÂ±u,n â€”5). Asterisk signifies value differs from BCAA-free diet group with P < 0.05.

Experiment 5 (table 6, fig. 2). Preliminaryexperiments were done with rats fed complete diet ad libitum to learn the relationships between concentration of tissue, concentration of KIC, duration of incubation,and rates of decarboxylation and transami-nation of KIC by liver, kidney and muscleslices. Results (fig. 2) showed that theratio of decarboxylation to transaminationin liver, kidney and muscle was about 2,0.2 and 0.003 respectively at all concentrations of KIC (0.25-2.5 HIM) and tissue(10-40 mg/0.5 ml medium) and at alltime periods (10-60 minutes) examined.The rates of both processes were proportional to KIC concentration in the range0.25 to 1.0 HIM and were constant from 1.5to 2.5 mM. Both rates were linearly proportional to tissue concentration in therange 10 to 40 mg/0.5 ml and to durationof incubation up to 40 minutes. For ex-Seriment 5, we selected the following con-

itions: KIC concentration of 0.25 and 2.0mM; tissue concentration, 10 mg/0.5 mlmedium; duration of incubation, 20 minutes. Results, expressed as /Â¿mole14CO2and 14C-leucine produced from [1-14C]KICper g tissue (wet weight) per hour, areshown in table 6. Rats fed complete dietminus BCAA, or this diet containing 1 or 2times the MDR of BCAA, showed no significant difference in rates of decarboxylation or transamination by the three tissues.In contrast, addition of 1 or 2 times theMDR of BCKA to the BCAA-free diet

caused a 2 to 6 times increase in the decarboxylation of [1-14C]KIC by the liverslices. Transamination of KIC by liver wasunaffected. The dietary BCKA did not de-tectably influence either decarboxylationor transamination of KIC by kidney ormuscle slices.

DISCUSSIONThe present experiments confirm Wol-

hueter and Harper (12) in showing thataddition of 3 times the MDR of leucine, or

20 30 Â«Or-i MUSCLE S.'C[

Fig. 2 Preliminary phase of experiment 5.This figure shows the relations between o-keto-isocaproic acid (KIC) metabolism to CO2 or leucine and: KIC concentration in medium (A andB), mg tissue slice/0.5 ml medium (C and D),and duration of incubation (E and F). Verticalscale shows dpm of "COa or [l-MC]leucine produced per incubation vessel from [1-"C]KIC.Values are mean Â±SE ( n = 4 ).



2 times the MDR of leucine, valine andisoleucine, causes a 20 c/c to 30% increasein the specific activity and total amount ofbranched-chain keto acid dehydrogenasein rat liver. In addition, our results showthat BCKA have the same influence, butare 2 to 6 times more potent than theBCAA in this respect.

The effect of BCAA and BCKA on activities of branched-chain dehydrogenase isspecific both for organ and enzyme. Thechange occurs in liver but not in kidney;BCKA dehydrogenases are affected, butpyruvate and a-ketoglutarate dehydrogenases are not. This result is consistent withthe fact that the latter two a-keto acids areoxidatively decarboxylated by specific pyruvate and a-ketoglutarate dehydrogenases(15), while the three BCKA are probablydegraded by one branched-chain keto aciddehydrogenase (16). Some authors havesuggested that in bovine liver two BCKAdehydrogenases are present: one specificfor the decarboxylation of KIC and KMVand the other specific for KIV (17, 18).However, our study in the rat confirmsWohlhueter and Harper's results (12) and

shows a coinduction of activity for allthree BCKA when any one of the threeBCAA or any one of the BCKA is addedto the diet at sufficiently high level. Thisis compatible with a single hepatic enzymespecific for decarboxylation of KIV, KICand KMV.

The strongest effect in the experimentswith tissue homogenates was producedwhen all three BCKA were fed at molarlevels corresponding to two times theMDR of BCAA and amounted to a 2 to 3fold increase in hepatic specific and totalactivities. Simultaneously kidney branched-chain dehydrogenase activity declined by40c/c, but since 90% of the branched-chaindehydrogenase is located in the rat liver(12), the overall effect was about adoubling in the capacity of the rat to de-carboxylate BCKA.

The findings with tissue homogenateswere confirmed and extended by thosewith tissue slices. The major pathway forKIC in liver slices is decarboxylation, andin kidney and muscle slices is transamina-tion. Addition of 1 or 2 times the MDR ofBCAA to the BCAA-free diet had little orno effect on the rates of these processes.

But the addition of the molar equivalentamounts of BCKA to the same diet causeda 2 to 6-fold acceleration of decarboxylation of KIC by liver slices; meanwhiletransamination of KIC by liver, and bothdecarboxylation and transamination of theketo acid by kidney and muscle, remainedunchanged. These findings suggest thataddition of BCKA to the diet of rats causesa specific alteration in the rate of oxidationof these acids in the liver, other aspects ofBCKA metabolism remaining relativelyunchanged.

What is the mechanism for the effect onhepatic branched-chain dehydrogenase?After the BCKA are ingested, the livercells are exposed to a high concentrationof these substances in the portal blood.The resulting increase in hepatic branched-chain dehydrogenase, for which BCKA arethe substrates, could result from an increase in synthesis of the enzyme, decreasein its rate of degradation, change in itsconformation or from activation of an inactive precursor.

The present findings on stimulation ofthe hepatic enzyme by BCKA may explainthe low nutritional efficiency of BCKAcompared to a-keto derivatives of methio-nine and phenylalanine (10, 11). IngestedBCKA are transported to the liver via theportal vein. In this organ, the rate of decarboxylation is about 2 times greater thanthat of transamination. Only those BCKAmolecules which escape decarboxylation inthe liver can reach the highly active amino-transferases in kidney and muscle (wherethe ratio of decarboxylation to transamination is about 0.2 and 0.03 respectively) andbe converted to the corresponding BCAA.As the dietary intake of BCKA is raised,progressive increase in hepatic branched-chain dehydrogenase activity and contentwill increase the proportion of ingestedBCKA which is degraded in the liver andreduce the proportion which is convertedto BCAA in muscle and kidney. Thus thestimulation of the hepatic enzyme byBCKA may explain why the molar dietaryrequirement for KIV and KIC to achievemaximal growth is 3 to 4 times greaterthan for valine and leucine respectively(2, 9).

Goldberg and Odessey ( 19) observedthat when [l-14C]leucine was incubated



with tissue slices from the fed rat, the rateof formation of 14COL.per g of tissue wassimilar for liver and muscle. Previous ( 12 )and present studies in the fed rat indicatethat the rate of conversion of [l-14C]leu-cine to 14CO2 will be determined in liverby the relatively slow step of transamina-tion, and in muscle by the relatively slowprocess of decarboxylation; and that eventhough the oxidation of 1-C of leucine toCOj is equally rapid in liver and muscle offed rats, the oxidation of 1-C of KIC isabout 15 times more rapid in the formerthan the latter tissue. With fasted rats, therate of 14COL,production from [I-14C]leu-cine was 5 times greater by muscle thanby liver, perhaps because of an increase inbranched-chain dehydrogenase activity instarving muscle (19). Goldberg and Odes-sey concluded that substantial amounts ofendogenous BCAA can be oxidized (viaBCKA as an intermediate) in muscles ofthe fasting rat ( 19). This conclusion doesnot conflict with the view (rÃ©f.12 and current study) that in the fed rat, the majorsite for decarboxylation of exogenous (dietary) BCKA is in the liver.

The current findings on the stimulationof hepatic branched-chain keto acid dehydrogenase by dietary BCKA in the ratshould not be directly extrapolated toman. It has been shown (20) that theorgan distribution of branched-chain dehydrogenase activity differs significantlybetween rat and man. Whereas in the ratthis enzyme is essentially hepatic, in man70% of the activity is extranepatic. Furthermore, in cirrhotic human liver thespecific activity of the dehydrogenase is10^ to 60% lower than that in normalhuman liver. These comparisons suggestthat the nutritional efficiency of BCKAmay differ considerably between normalrat, normal man and cirrhotic man.

LITERATURE CITED1. Wood, J. L. & Cooley, S. L. (1954) Sub

stitution of a-keto acids for five amino acidsessential for growth of the rat. Proc. Soc.Exp. Biol. Med. 85, 409-411.

2. Chawla, R. K. & Rudman, D. (1974)Utilization of Â«-keto and a-hydroxy analogues of valine by the growing rat. J. Clin.Invest. 54, 271-277.

3. Meister, A. (1965) Biochemistry of theamino acids. Vol. 1. Academic Press, Inc.,New York. 2nd Ed. p. 347.

4. Rudman, D. (1971) Capacity of humansubjects to utilize keto analogues of valineand phenylalanine. J. Clin. Invest. 50, 90-96.

5. Walser, M., Coulter, A. W., Dighe, S. &Crantz, F. R. (1973) The effect of ketoanalogues of essential amino acids in severechronic uremia. J .Clin. Invest. 52, 678-690.

6. Close, J. H. (1974) The use of amino acidprecursors in nitrogen-accumulation diseases.N. Eng. J. Med. 290, 663-666.

7. Richards, P., Brown, C. L., Houghton, B. J.& Thompson, E. (1971) Synthesis ofphenylalanine and valine by healthy anduremie man. Lancet 2, 128-134.

8. Rogers, Q. R. & Harper, A. E. (1965)Amino acid diets and maximal growth in therat. J. Nutr. 87, 267-273.

9. Chawla, R. K., Stackhouse, W. J. & Wads-worth, A. D. (1975) Efficiency of a-keto-isocaproic acid as a substitute for leucine indiet of the growing rat. J. Nutr. 105, 798-803.

10. Chow, K-W. & Walser, M. (1974) Substitution of five essential amino acids by theiralpha-keto analogues in the diet of rats. J.Nutr. 104, 1208-1214.

11. Gaby, A. R. & Chawla, R. K. (1976) Efficiency of phenylpyruvic and phenyllacticacids as substitutes for phenylalanine in thediet of the growing rat. J. Nutr. Ã•06,158-168.

12. Wohlhueter, R. M. & Harper, A. E. (1970)Coinduction of rat liver branched chaina-keto acid dehydrogenase activity. J. Biol.Chem. 245, 2391-2401.

13. Weyland, F., Steglich, W. & Tanner, M.( 1962) Eine neue mÃ©thodezur Umwandlung von a-aminosÃ¤uren in a-ketosÃ¤uren.Justus Liebig's Ann. Chem. 658, 128-150.

14. Mendenhall, W. M. (1971) Introductionto Probability and Statistics. 3rd Ed., Wads-worth Publishing Co., Belmont, Calif.

15. Reed, L. J. & Cox, D. J. (1970) The Enzymes. (Boyer, P. D., ed.), Vol. 1, AcademicPress, New York, N.Y., pp. 213-237.

16. Dancis, J. (1959) Phenylketonuria andmaple sugar urine disease. Bull. N.Y. Acad.Med. 35, 427-432.

17. Connelly, J. L., Danner, D. J. & Bowden, J.A. (1968) I. Branched chain o-keto acidmetabolism. Isolation, purification and partialcharacterization of bovine liver a-keto iso-caproic, a-keto-0-methyl valeric acid dehydrogenase. J. Biol. Chem. 243, 1198-1203.

18. Bowden, J. A. & Connelly, J. L. (1968)Branched chain a-keto acid metabolism. II.Evidence for the common identity of o-keto-isocaproic acid and a-keto-/3-methylvalericacid dehydrogenase. J. Biol. Chem. 243, 3526.

19. Goldberg, A. L. & Odessey, R. (1972) Oxidation of amino acids by diaphragms fromfed and fasted rats. Am. J. Physiol. 223,1384-1391.

20. Khatra, B. S., Chawla, R. K., Sewell, C. W.& Rudman, D. (1977) Distribution ofbranched chain a-keto acid dehydrogenase inprimate tissues. J. Clin. Invest. 59, 558-564.


effect of dietary branched-chain /-reto acids on hepatic branched

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