Pamela L Crowell, MDaudNawabi,

Kevin Maria

P Block, Joyce J Repa, Nimbe G Buse, andAlfredE Harper

Torres,

ABSTRACT Diets containing high quantities of individual branched-chain a-keto acids (BCKAs) or a combination of BCKAs as used for treatment of renal disease were fed to rats. When the diet contained a single BCKA, its concentration was high

in

plasma

amino

acid

was

and

the

high

in plasma

concentration

of

and

its

brain.

corresponding

Liver

BCKA

dehy-

drogenase (BCKD) was 42% active in control rats. Consumption ofdiets containing 0.38 mol/kg diet of ct-ketoisocaproate (KIC), a-keto-3-methylvalerate (KMV), or ct-ketoisovalerate (KIV) resulted in complete activation ofliver BCKD. Consumption of the diet containing the combination of BCKAs increased basal BCKD activity ofliver twofold. Muscle BCKD was activated after feeding the KIV diet (2-fold), the KIC diet (3-fold), and the KMV diet ( 15-fold). Total BCKD activity

of liver

ments.

Activation

is consistent

nase,

muscle

ofliver

with

was

unaffected

their

dietary

treat-

by dietary BCKA BCKD kinase in

1990;52:313-9.

WORDS

Branched-chain

branched-chain

a-keto

cv-ketoisocaproate,

by

and muscle BCKD ability to inhibit

AmfClinNutr

vitro.

KEY

and

ct-keto

acids,

amino

acid

dehydroge-

acid concentrations,

L-al/oisoleucine

cine,

and

valine,

have

of several amino acids, amino acids (BCAAs) been

used

especially leucine,

as therapeutic

those isoleu-

agents

in the

treatment ofdiseases that result in protein intolerance or negative nitrogen balance, such as chronic renal failure ( 1 ), hepatic encephalopathy (2), or postoperative nitrogen wasting (3). Improvement in nitrogen balance with keto acid therapy is due partly

to conservation

the keto

acids,

possibly

also

of nitrogen

reducing

a-ketoisocaproate,

through

transamination

the loss through

urea

of proteolysis

of tissue

to inhibition

the a-keto

analog

ofleucine

regulated in part by a phosphorylation phosphorylation (activation) mechanism chain

kinase a-keto

Am J C/in Nuir

and

phosphatase,

acids

(BCKAs)

1990;52:3

13-9.

the

Printed

synthesis,

substrates in USA.

(6).

by

(BCKD), catabolism,

The

the is

and deby an inbranched-

for BCKD © 1990 American

inhibit

BCKD

kinase

activity

in

vitro

(7,

8),

ity (12).

Thus,

administration

ofbranched-chain

amino

or keto

acids by diet or infusion may increase catabolism ofthese cornpounds in vivo. An excess ofleucine, either fed or injected, induces an antagonism in rats fed a bow-protein diet (1 3, 14). In this condition the plasma and muscle concentrations of leucine increase greatly and those of isoleucine and valine decline. Changes in the concentrations ofthe a-keto analogs ofleucine, isoleucine, and valine follow the same pattern as those oftheir corresponding amino acids. Food intake and growth are depressed; these effects are alleviated by the addition to the diet of small amounts of isoleucine and valine. BCAA oxidation increases whole

animal

(1 1), presumably

liver (9, 15) and muscle BCKD (10). To intakes ofthe branched-chain keto acids changes in BCAA metabolism as their acids when fed in the diet, we examined cesses of BCKAs on plasma and brain tions, plasma BCKA concentrations,

through

activation

of

establish whether high would cause the same corresponding amino effects of dietary examino acid concentraand liver and muscle

BCKD activities. Materials

and methods

Male Sprague-Dawley housed in individual,

rats, initially weighing 75-90 g, were suspended wire-mesh cages in a room lit

and

proteins

(inactivation) catalyzed

respectively are

of

(4, 5).

Branched-chain a-keto acid dehydrogenase rate-limiting enzyme in the pathway of BCAA

trinsic

addition,

thereby preventing inactivation of the enzyme. BCKD is activated in liver (9) and in muscle (10) in vivo by excesses of dietary leucine. Equimolar amounts ofdietary isoleucine and valine also activate liver BCKD (9), and supraphysiological amounts of injected isoleucine activate muscle BCKD (10). Whole-body oxidation of valine is stimulated by a dietary excess of leucine but not isoleucine ( 1 1 ). In studies done before the phosphorylation mechanism of BCKD activation was discovered, dietary BCKAs were shown to stimulate BCKD activ-

in the

Introduction The a-keto analogs of the branched-chain

in

and, Society

I From the Departments of Biochemistry and Nutritional Sciences, University of Wisconsin-Madison, and the Department of Medicine, Medical University ofSouth Carolina, Charleston. 2 Supported by NIH grants AM 10748 and BK-02001. 3 Address reprint requests to AE Harper, Department of Biochemistry, University ofWisconsin-Madison, 420 Henry Mall, Madison, WI 53706. Received June 26, 1989. Accepted for publication September 27, 1989.

for Clinical

Nutrition

313

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High branched-chain a-keto acid intake, branched-chain a-keto acid dehydrogenase activity, and plasma and brain amino acid and plasma keto acid concentrations in rats13

CROWELL

314

from 0700-1900. Rats were fed ad libitum a control diet with 90 g casein/kg diet supplemented with 3 g methionine/kg diet for 2 d (9). They were then trained to consume the control diet for 6 h/d, from 0900-I 500, for 10-12 d to synchronize the time of food consumption. Rats were then separated into groups of six animals

each

and

fed one

ofthe

following

diets

for 6 h/d,

as

above,

KIV/kg

diet

(combination

BCKA

diet).

In diets

2 (KIC),

3

(KMV), and 4 (KIV) keto acid concentration was 0.38 mob/kg diet [equimolar with the amount ofleucine, which is added to low-protein diets to induce the antagonism between leucine and both isoleucine and valine (13)]. The combination BCKA diet

contains

the

treatment

keto

acids

in proportion

of patients

with

for rats

chronic

renal

to those failure

used

(16).

in

Keto

(calcium salts) were added to the diet at the expense of carbohydrate. Body weight was monitored daily; food consumption was measured for 2 d before and the 2 d during the feeding of the experimental diets. All animals were cared for acids

in accordance complies with

with the University the principles stated

of Wisconsin in the 1985

policy, which NIH guide for

the care and use oflaboratory animals. Rats were killed by decapitation on the second day the experimental diets were fed, 3 h after the start ofthe feeding period, so that enzyme-activity measurements would be made during the absorptive phase. [Previous studies from our laboratory (17)

indicated

that

the maximal

response

ofliver

BCKD

in ani-

mals fed low protein diets occurs within 3 h of the start of the meal.] Mixed arterial-venous blood was collected from the neck

in heparinized

Livers

were excised,

beakers tively.

containing Basal (ie,

tubes

for

weighed,

amino

divided

and

keto

acid

analyses.

in half, and placed

ice-cold basal and total the in vivo phosphorybation

assay

in two

buffer, respecstate) and total

(fully activated, ie, dephosphorylated) liver BCKD activities were measured in separate homogenates as outlined by Block et al ( 18). Oastrocnemius and soleus muscles were isolated and frozen in liquid nitrogen for measurement of basal and total BCKD activity in muscle extracts (18). For both liver and muscle, basal homogenizing media contained inhibitors of BCKD phosphatase

(potassium

isocaproate for liver press interconversion Complete activation plished

tracts

fluoride)

by preincubation

of liver

at 37 #{176}C in buffer

assay, assay

a-chloroisocaproate media for total

and

BCKD

kinase

(a-chloro-

and dichloroacetate for muscle) to supofthe complex during tissue preparation. of BCKD for total assays was accomhomogenates

containing

assays;

magnesium.

or muscle

ex-

In the

liver

was present in homogenizing 50 mmol potassium fluoride/L

and was

in the assay medium only so that the assay media for basal and total BCKD would be identical. Liver and muscle BCKD assays were optimized for each tissue and hence different methods were used. Activity measurements are therefore included

not

directly

Amino

comparable

acid

between

concentrations

the two

were

tissues.

measured

brain as described by Tews et al (19) with dinopropionic acid as the internal standard.

in plasma

and

L-a-amino-/3-guaniL-tryptophan

mea-

surements were obtained by using a fluorometric assay plasma samples (19) and a high-performance-liquid-chromotography assay for brain samples (20). Concentrations plasma BCKAs were measured by gas-liquid chromatography

for

as described

et al (2 1). Alpha-ketovalerate

was

the

nificant (23).

The calcium salts of the BCKAs (used in the diets) were a kind gift from Mackenzie Walser ofiohns Hopkins University School ofMedicine in Baltimore. Alpha-chboroisocaproate was a gift from Ri Strohscheim and R Simpson of Sandoz, East Hanover, NJ. The sodium salts ofthe BCKAs (for standards in gas chromatography), a-ketovalerate and L-alloisoleucine (Lall-lIe) were from Sigma Chemical Co, St Louis. L-[b-’4C]valine was from Amersham, Arlington Heights, IL. All other materials were from commercial sources and were ofreagent grade or better.

Results Food

intake

After rats had been trained to consume the casein control diet for 6 h/d for 10-12 d, groups ofsix rats each were fed one of the five diets (control, KIC, KMV, KIV, or combination BCKA diet). On the first day they were fed the experimental diets, only the group consuming the KIC diet ate less food than the controls (data not shown). On the second day, before the animals were killed, the 3-h food intakes ofthe groups consuming the KIC, KMV, or KIV diets were significantly depressed to 65.4%, 8 1.6%, and 69.5%, respectively, ofthe control value (6.8 g). Food intake of the group fed the combination BCKA diet was not depressed. Body weights were the same in all groups (data not shown). Plasma

keto acid concentrations

Concentrations ofpbasma BCKAs in rats fed the casein control diet were 9.8-1 3.2 jmob/L (Table I). Consumption of the KIC diet resulted in a KIC concentration sixfold higher than the control and concentrations of KMV and KIV at the limit of detection (< 2 tmol/L). In rats consuming the KMV diet, plasma KMV concentrations were 40-fold higher than in controls; KIC and KIV concentrations decreased to < 3 mol/L. In rats consuming the KIV diet, KIV concentrations were 17fold higher than in controls; KIC and KMV concentrations did not change significantly. Consumption of the combination BCKA diet (KIC + KMV + KIV) resulted in two- and threefold increases in KIC and KMV concentrations, respectively, but no significant change in KIV. The sum of the concentrations ofthe three BCKAs was highest in the group fed the KMV diet,

followed

by those

and the control Plasma

amino

fed the

KIV,

KIC,

BCKA

combination,

diet. acid concentrations

Concentrations of plasma amino acids of rats fed BCKA diets for 2 d are summarized in Table 2. Rats fed the KIC (ketoleucine) diet had higher Leu and lower lIe and Val concentrations

of

by Crowell

internal standard. [l-’4C]a-ketoisovalerate was synthesized from L-[ l-’4Cjvaline by the method ofRudiger et al (22). Values are presented as means ± SEM with six rats per group. Analysis of variance was by the Newman-Kuehls test for differences among groups. Student’s I test was used to test differences between two groups, with p < 0.05 considered sig-

than

did

rats

fed the

control

diet.

Similarly,

lie was

higher

in rats fed the KMV (ketoisoleucine) diet than in those fed the control diet, and Leu and Val concentrations were lower. The

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for 2 d: control; control + 57 g a-ketoisocaproate/kg diet (KIC diet); control + 57 g R,S,-a-keto-f3-methylvalerate/ kg diet (KMV diet); control + 52 g a-ketoisovalerate/kg diet (KIV diet); or control + 5.34 g KIC + 5.0 g KMV + 4.34 g

ET AL

DIETARY

KETO

ACIDS

AND

BCKD

315

ACTIVITY

TABLE

1 Concentrations

ofplasma

branched-chain

a-keto

acids (BCKAs)

in rats fed keto acid diets* Diet

BCKAs

Control

(n

=

KIC (n

6)

=

6)

KMV

(n

KIV (n

6)

=

Combination (n = 6)

5)

=

13.2 ± l.8a l2.3±4.0a 9.82 ± l.09a 35.3 ± 5.9a

KIC KMV KIV Sum ofBCKAs

i± SEM. Data were transformed

U

p