Clinical Science (1983) 64,s 17-526
5 17
Effects of ingested steak and infused leucine on forelimb
metabolism in man and the fate of the carbon skeletons and
amino groups of branched-chain amino acids
MARINOS ELIA* AND GEOFFREY LIVESEYt
Metabolic Research Laboratory. N w e l d Department of Clinical Medicine, Radclire Injrmary. Odord,
and N w e l d Orthopaedic Centre, Odord, U.K.
(Received I2 July128 September 1982; accepted I7 November 1982)
Summary
1. The effects of ingested grilled beef steak
(250 g raw weight of lean meat) and infusion of
leucine (3-8 g) on human forelimb metabolism
were studied by monitoring the concentrations of
various metabolites in arterial (A) and venous (V)
blood of four overnight fasted and rested men.
2. The mean basal A-V for branched-chain
2-0x0 acid (BCOA) was small (-3.6 pmolll).
After ingestion of steak or administration of
leucine there were large positive increases in the
A-V for branched-chain amino acid (BCAA) but
increase in the negative A-V for BCOA was
relatively small.
3. Within 2 h of ingestion of steak, BCAA
accounted for approx. 50% of those amino acids
with a positive A-V and glutamine for up to 75%
of those with a negative A-V; the negative A-V
for alanine decreased to 10% of its basal value.
Infusion of leucine produced a large positive A-V
for leucine by forelimb, a doubling in the negative
A-V for glutamine and a rise in the blood
glutamine concentration; the negative A-V for
alanine was virtually unchanged and the blood
alanine concentration showed a late significant
decrease.
4. After ingestion of steak there was a two- to
three-fold rise in the arterial insulin concen-
* Present address: MRC Dunn Nutrition Centre,
New Addenbrookes Hospital, Trumpington Street,
Cambridge CB2 lQE,U.K.
Present address: ARC Food Research Institute,
Colney Lane, Norwich NR4 7UA, U.K.
Correspondence: Dr G. Livesey, ARC Food
Research Institute, Colney Lane, Nonvich NR4 7UA,
U.K.
tration, little change in the positive A-V for
glucose and a decreased negative A-V for
‘glycolytic products’ (alanine + lactate + pyruvate), suggesting increased utilization of glucose
carbon. Infusion of leucine doubled the arterial
insulin concentration; the A-V for glucose
decreased, that for lactate, pyruvate and alanine
remained unchanged, suggesting decreased
utilization of glucose carbon.
5. Circulating BCOA was distributed almost
entirely in the plasma space.
6. In a variety of clinical conditions (insulindependent diabetes, cirrhosis, muscular dystrophy and starvation), the basal plasma concentrations of BCOA correlated well with those
of BCAA (r = 0.989). Infusion of leucine
increased the plasma BCAAIBCOA ratio to the
same extent (about 40%) in each clinical condition despite considerable variations in the rate
of leucine clearance.
7. The observations indicate that both ingested
steak and infused leucine produce important
changes in the selection of respiratory fuels by the
human forelimb, that BCOA is preferentially
oxidized rather than released from human limb
tissues, and that glutamine, not alanine, is the
major amino group carrier leaving the forelimb
both after a protein meal and after leucine
administration. Changes in cellular uptake or
transamination of leucine appear not to be
responsible for the varied rates of leucine
clearance in a variety of clinical conditions.
Key words: alanine, branched-chain amino acids,
branched-chain 2-0x0 acids, glutamine, leucine,
respiratory fuel selection.
0143-522 1/83/0505 17-10%2.O0 @ 1983 The Biochemical Society and the Medical Research Society
518
M . Elia and G. Livesey
Abbreviations: BCAA, branched-chain amino
acid; BCOA, branched-chain 2-0x0 acid.
Introduction
Peripheral tissues, skeletal muscle in particular,
are considered to be major sites for the transamination of the branched-chain amino acids
(BCAA), leucine, valine and isoleucine [l, 21.
Branched-chain 2-0x0 acids (BCOA), however,
may either be oxidized in muscle, as indicated by
experiments in vitro [3,4], or be released into the
circulation for removal by the liver, as shown in
the rat hind limb in vivo 151. Whether human limb
tissues also release BCOA into the circulation is
investigated here together with the relationship
between plasma BCAA and BCOA concentrations in several conditions (diabetes, starvation, cirrhosis and muscular dystrophy) known
to be associated with altered metabolism of
BCAA [61.
Increased metabolism of BCAA by rat muscle
in vitro is associated with increased release of
alanine [7-9] and glutamine [10-11]. Alanine
and glutamine are quantitatively the most important amino group carriers leaving the human
forelimb in vivo [ 121, and a leucine meal has been
shown to increase glutamine and total nitrogen
release from forearm muscle 1131. There is,
however, still doubt about which of these amino
acids is quantitatively the more important amino
group carrier leaving the human forelimb in vivo
under conditions of increased BCAA utilization.
Therefore we have measured the arteriovenous
concentration differences of various amino acids
across the human forelimb after giving a protein
meal and intravenous leucine to normal man.
Measurement of glutamine concentrations with
a reliable sensitive enzymatic method soon after
obtaining samples is particularly important for
this work. Estimations with an amino acid
analyser can be unsatisfactory since glutamine
may not separate from other amino acids, as in
the study of Wahren et al. [141, and because
glutamine degrades spontaneously during storage
in frozen solution [151 and during passage
through the hot column of an analyser.
Methods
values f SEM), who had previously been on
normal diets.
Plasma for the determination of antecubital
venous branched-chain amino and 2-0x0 acids in
patients with a variety of clinical conditions (Fig.
1) was taken between 07.30 and 09.00 hours from
overnight-rested subjects who had also fasted
either overnight or longer (starved subjects). The
13 normal subjects (1 1 males, two females, age
29 f 3 years, weight 69 f 2 kg, height 174 f 1
cm) had previously been on normal diets which
were estimated to supply about 100 g of
protein/day. The seven cirrhotic patients (two
males, five females, age 50 2 years, weight 59 f
5 kg, height 171 f 3 cm: four alcoholic cirrhosis,
two primary biliary cirrhosis, one chronic active
hepatitis on 10 mg of prednisolone/day), all had
well compensated liver disease with a mean
plasma albumin of 35 g/l, and had previously
been on hospital diets which supplied 40-60 g of
proteidday. The four muscular dystrophy
patients (three males, one female, age 21 f 5
years, weight 47 f 12 kg, height 154 ? 9 cm: two
Duchenne muscular dystrophy with one judged
histologically to be pseudohypertrophic, one
facioscapulohumeral muscular dystrophy, one of
unknown cause) had previously been on hospital
diets which supplied 60-80 g of proteidday and
all had evidence of some muscular atrophy. The
nine insulin-dependent diabetic subjects, in addition to overnight resting and fasting, had
plasma sampled before receiving morning insulin;
these subjects (seven males, two females, age 42
f 6 years, weight 65 f 4 kg, height 172 k 2 cm)
had previously been on a variable carbohydrate
hospital diet supplying approximately 100 g of
proteidday. The four subjects who starved for 2
and 4 days (four males, age 30 f 2 years, weight
76 kg, height 172 f 2 cm) had previously been on
normal diets estimated to supply approximately
100 g of protein/day and returned to similar diets
on day 5 after the start of the 4 day fast. Leucine
infusions were performed in four subjects from
four of the above groups: cirrhosis, diabetes,
muscular dystrophy and starvation (before and
on day 4 of starvation). These 16 individuals had
only venous blood withdrawn, for analysis of
plasma BCAA and BCOA concentrations, and
were included in a previous study [61 which gives
their individual details.
Experimental subjects
Experiments on the ingestion of steak and the
infusion of leucine were started between 08.00
and 09.00 hours on four overnight-rested and
fasted normal male subjects (age 30 f 8 years,
weight 69 f 2 kg, height 174 f 2 cm; mean
Procedures
Analysis of blood metabolites.
(or plasma prepared immediately
were deproteinized with 2 vol.
perchloric acid. After removal
Blood samples
after sampling)
of 10% (w/v)
of the protein
Branched-chain amino acid metabolism
precipitate, the extracts were neutralized with
20% (w/v) KOH, then either kept on ice or stored
at -2OOC until analysed. The concentrations of
BCOA (sum of 4-methyl-2-oxovalerate, 3-methyl-2-oxobutyrate and 3-methyl-2-oxovalerate)
were determined by using leucine dehydrogenase
purified from Bacillus subtilis and corrected, in
the case of blood, for a 77% recovery [51.
D-Glucose, L-lactate, pyruvate, acetoacetate and
D-3-hydroxybutyrate were determined by
methods described elsewhere [ 161. L-Alanine was
determined with L-alanine dehydrogenase [ 171.
L-Glutamate was measured by the method of
Bernt & Bergmeyer [ 181 as modified by Cornell et
al. [191 and L-glutamine by the glutaminase
method [201. Other amino acids were determined
on a Jeol JLCdAH auto-analyser. BCAA concentrations are the sum of leucine, isoleucine and
valine determined on the analyser. Pyruvate,
acetoacetate, L-glutamate and L-glutamine were
assayed within a few hours of withdrawing blood.
Insulin was measured by the double antibody
technique of Albano et al. [211.
Experiments with radiolabelled substrates. The
proportion of plasma to whole blood volume was
determined with inulin [l-14Clcarboxylic acid, a
marker of the extracellular space. The distribution of BCOA in blood was determined in two
ways: enzymatic assay of whole blood and
plasma BCOA and recovery in plasma of the
tracer 4-methyl-2-oxo[l-14Clvalerate, prepared
from ~-[l-~~Clleucine
1221, after mixing with
whole blood. The radiochemicals were from The
Radiochemical Centre, Amersham, Bucks, U.K.
Arteriovenous
concentration
dverence
measurements. Samples of blood were removed
simultaneously from the brachial artery (A) and
the antecubital vein (V). These samples were
withdrawn via flexible lines connected to Teflon
catheters fixed in position for 30 rnin before the
first basal samples were taken. The venous
catheters were directed for 3 4 cm into the deep
vein with intent to increase the contribution of
blood draining muscle. The hand was not
occluded during arteriovenous blood sampling.
Blood was also sampled simultaneously, for
comparison, from a superficial mid-forearm vein
(SV)via a flexible connection to a venous needle.
A and V blood samples were taken in duplicate, SV samples singly and basal samples were
taken at two different times. Only whole blood
was used for A-V difference measurements.
Blood flow rates were not measured; previous
studies have not demonstrated any significant
change in forearm blood flow after ingestion of
steak or administration of leucine [13,14,231.
Protein meal. Grilled lean beef steak (250 g
5 19
raw weight) was ingested, over a period of 10-12
min, starting at time 0 min. Arterial and venous
blood samples were taken in duplicate simultaneously to obtain basal samples at -30 and 0
min, and thereafter at 30 min intervals for the
next 3 h. All blood samples (3 ml each) were
deproteinized immediately. Arterial plasma for
insulin determination was also taken at these
times.
Infusion of leucine. L-Leucine [British Drug
Houses Ltd; 3.8 g (29 mmol) dissolved in 200 ml
of sodium chloride solution, 154 mmolh (saline)]
was infused over a period of 8-10 min into the
antecubital vein starting at time 0 min. Arterial
and venous blood samples were taken simultaneously in duplicates at both -15 and 0 rnin to
obtain eight basal samples from each subject and
thereafter in duplicate at + 15, +30 and +60 min.
Plasma for insulin determination was also obtained at these times.
Ambient temperature. All experiments involving protein meals and leucine infusions were
performed at ambient temperatures of 19-21 OC;
skin temperatures were not measured. Venous
plasma from patients with a variety of clinical
conditions (Fig. 1) was taken at hospital ward
temperatures of approx. 2OoC, though temperatures were not measured on all occasions.
Statistics. Data were anlysed by using the
Students t-test, which was applied to paired
observations where possible.
Results
Distribution of BCOA in blood
The basal concentration of BCOA in forearm
venous plasma was 69 & 4 pmolh (mean f SEM)
and that in venous whole blood was 43 f 3
pmol/l. Plasma accounted for 60.7 & 1.0% of the
whole blood volume, so that 97% of the BCOA
present in the whole blood was accounted for by
that in plasma. This distribution agrees closely
with an observed 95 & 3% (mean f SEM, n = 8)
recovery of tracer quantities of 4-methyl-2-0~0[lJ4C1valeric acid in plasma after being mixed
with whole blood for 15 min at 37OC under CO,
+ 0, (5 :95, v/v).
Basal plasma concentrations of BCAA and
BCOA
Venous plasma concentrations of BCAA and
BCOA in the various clinical conditions are
shown in Fig. 1. In the normal subjects (n = 13)
the basal BCAA concentration was 410 f 10
pmol/l (mean f SEM) compared with 69 f. 4
pmol/l for the BCOA concentration. During a
M.Elia and G. Liuesey
520
4-day fast the BCAA concentration increased
rapidly to reach peak concentrations on day 2;
within 24 h of refeeding on day 5 the BCAA
concentrations returned to normal (see reference
[6]). By contrast the BCAA/BCOA concentration ratio varied little throughout the period of
study.
In patients with liver disease the venous plasma
concentrations of both BCAA and BCOA were
decreased significantly, but in the muscular
dystrophy patients these values were not significantly different from normal. The venous
plasma concentrations of BCAA and BCOA in
the diabetic patients were somewhat variable.
When these patients were arbitrarily divided into
two groups, on the basis of their fasting blood
glucose being less or greater than 10 mmol/l, the
group with the higher blood glucose (14.9 f 1.0
mmol/l, mean f SEM,n = 4) showed significantly
elevated concentrations of both BCAA and
BCOA (593
48 and 97 f 9 ,umol/l respectively), while those with the lower, more normal
blood glucose (7.1 f 1.0 mmol/l, n = 5) showed
concentrations of BCAA and BCOA (429 f 45
and 59 f 5 pmol/l respectively) not significantly
different from normal.
The close relationship (correlation coefficient, r
= 0.989) between the venous plasma concentrations of BCAA and BCOA as seen in the
post-absorptive subjects in the various clinical
conditions (Fig. 1) was not observed after leucine
infusion or after protein feeding (see below). In
normal subjects given leucine there were large
increases in the circulating concentrations of both
plasma and whole blood BCAA (due almost
entirely to leucine; Table l), and BCOA (Table
2). Similar changes in plasma BCAA and BCOA
were observed after infusion of leucine into the
cirrhotic and insulin-dependent diabetic subjects
and the normal subjects after 4 days of starvation
(data not shown; see reference 161). In the
muscular dystrophy patients the increments in
BCAA concentrations were somewhat greater
than in the other groups of subjects, owing to the
TABLE
1. Arterial (A) and arteriovenous ( A - V ) concentrations of amino acids in blood across the
forelimb offour normal subjects given intravenous leucine
L-Leucine (3.8 g) was infused between 0 and 10 min. Values (in pmol/l, or munits/l for insulin) are
means k 1 SEM (four subjects). Results significantly different from the mean of pre-infusion (basal)
measurements (four subjects): *P < 0.05; **P< 0.01.
Time after
Concentrations
lriirinr infiirinn
(min)
Tau
Glu
...
A
A
A-V
Gln
A
G~Y
A-V
A
A-V
Ala
A
A-V
Val
A
lleu
A-V
A
A-V
Leu
A
TYr
Phe
Om
LY s
His
Try
A%
Insulin
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
Basal
288 f 1
153 f 8
+15+4
544 f 19
-29 f 6
340 f 37
-5 f 9
248 f 18
-42 f 9
212f I
+5 f 5
72 f 7
+4 f 3
126 f I 1
-4 f 4
55f4
-I f 1
54 f 8
+7 f 7
102 f 13
-52 I 1
187 ? 20
-11 f 4
118f23
-5 f 9
96 f 10
-2f I I
82 5
+4+5
4f I
+
30
15
+
218 16
159 f 5
+21 f 3
567 f 20
-59 f 12'
324 f 38
-7 f 3
270 f 23
-37 f 7
218f 8
-5
3
9 9 f 13'
+9 f 10
I190 f 62.'
+247 f 42''
54f I I
--I f 2
51f8
-25 1
81 f 13
-13f4
199 f 27
-24 f 8
93 f 24
- 3 f 18
7 5 f I2
+
+5 f 5
87 f 7
+4 f 8
7+1
217 f 22
154 f 5
+i4 f 5
589 f 13'
-59 f 17'
330 f 33
+5
k 16
253 f 21
-45 f I5
191 f I5
-8 f 7'
72 f 8
-6f6
700 f 53+57 f 24'
5 9 k 18
+4 f 8
44 f 7
If7
92 f 10
-1+3
199f 19
-26 f 4'
120 f 34
-12 f 22
90 f 6
+7f I1
81 f 10
-11 f 5
7f2
60
152f 10
+I1 f 3
579 f 8'
-57f 17
331 f 42
-I0 f 14
228 f 19'
-42 2 17
186 f 23
+ 7 f 13
47 f 5"
-10 f 6
432 f 30"
-3 f LO
39 f '6
-1 f 11
33 f 2'
-3f I
98 f 7
+5 f 3
208 f 22
-3 f 10
140 f 26
+l9f22
8 5 f 17
-2 f 29
101 f 20
+ 8 f 16
4+ 1
52 1
Branched-chain amino acid metabolism
TABLE2. Arterial (A), arteriovenous (A-V) concentrations of branched-chain amino acids (BCAA) and branched-chain
2-0x0 acids (BCOA) in blood infour normal subjects q/?erprotein (steak) ingestion and leucine administration
Values in pmol/l are means & 1 SEM (four subjects). Results significantly different from the pre-ingestion or pre-infusion
(basal) measurements (four subjects): *P < 0.05; **P< 0.01.
Time after
(min)
BCAA
Amino acid concentrations (pmolA)
...
Basal
A
A-V
A
A-V
BCOA
BCAA
BCOA
30
60
329 f 10
449 f 37
-12 f 3
+I2 f 7*
54.4 f 7.3 52.5 f 8.5
-1.9 f 1.4 -2.4 f 2.3
90
120
636 f 42** 775 f 43.
+81 f 15..
+88 f 27.
58.5 f 9.0
59.5 f 8.5
-5.1 f 1.5.
-5.9 f 1.4.
Time aRer
leucine infusion?
(min) ...
Basal
15
A
A-V
A
A-V
409 f 17
+5 f 7
42.8 f 2.0
-5.5 f 3.8
1506 f 66***
+250 f 49**
113.0 f 7.0**
+2*8f 6
853 f 55**
+88f 30.
61.4 k 8.0'
-5.1 f 1.4.
150
180
794 f 39**
+23 k 24
57.5 f 5.0
-3.9 f 0.8
786 87*
+I2 k 30
53.6 f 6.8
-2.3 f 0.9
Amino acid concentrations(pmol/l)
60
30
666 f 55..
-6 f 13
61.7 k 3.3.
-14.2f 3.6.
963 f '
.
1
7
+42 f 28
86.9 f 3.9..
-9.4 f 2.4
Steak (250g raw weight) was ingested between 0 and 15 min.
t leuci cine (3.8 g) was infused between 0 and 10 min.
1OOO900
-
700 -
800
"
'1
l b l d glumsa
600-
:
-
4
-
z 10 mmoffll
12
-
0
'2
2
10-
d
-
400-
8
B-
300 -
0
500-
V
m
-
I2 h past-ingestion1
.
a
14
.
4
2
200
Cirrhosis In = 71
-
V
-
c4
6-
M
4-
a
-
NLXIIEI In = 41
Diabetic In = 41
Muscular dvavophv (n = 41
2-
Unksis(n=41
Suwation. day +4 (n = 41
BCOA (pmolh)
0-
FIG.1. Effects of various clinical conditions on the
plasma concentrations of branched-chain amino acids
(BCAA) and branched-chain 2-0x0 acids (BCOA).
Values (,umol/l) are mean concentrations & SEM;
vertical bars refer to the BCAA and horizontal bars to
the BCOA. The coefficient of correlation; r = 0-989.
Results significantly different from normal: *P 0.05;
**PO.Ol: ***P0.001.
FIG. 2. Effects of leucine infusion on the plasma
branched-chain amino acid (BCAA)/branched-chain
2-0x0 acid (BCOA) concentration ratio in various
clinical conditions. Values are mean ratios from four
subjects in each condition infused with L-leucine
(3-8 g), as described in the Methods section.
small body weight and leucine distribution
volume [61. However, in all the subjects given
leucine, the fractional rise in BCAA concentration was greater than that for BCOA so that at
15 and 30 min after the start of the leucine
infusion the BCAAIBCOA concentration ratio
increased by approximately 40% and returned
towards normal values by 60 min (Fig. 2).
M.Elia and G. Livesey
522
Effects of ingested steak and infused leucine
on the circulating insulin and amino acid
concentrations
After ingestion of steak there were significant
increases in the arterial concentrations of all the
measured amino acids except aspartate, tryptophan and taurine (Table 3). The BCAA were
amongst those showing the largest rise (leucine,
256%; isoleucine, 256%; valine, 182% of basal
values). The changes were accompanied by a twoto three-fold rise in the plasma insulin concentration (Table 3) and the substantial change in
the A-V values would suggest a temporary
change in amino acid exchange from net release
to net uptake (Fig. 3).
By contrast, leucine administration was fol-
lowed by significant increases in the arterial
concentrations of only leucine and glutamine
(Table 1). This was also associated with a twofold
rise in plasma insulin concentration (Table 1).
BCAA and BCOA exchange
Although the arterial concentration of BCAA
doubled after ingestion of steak, the concentration of BCOA increased by less than 20%
(Table 2). BCAA accounted for 50% of those
amino acids with a positive A-V between 1 and
2 h (Fig. 3). At the same time there was only a
small significant increase in the negative A-V for
BCOA from a basal value of -1.9 pmolll to a
value of -5 a 9 pmol/l at 2 h (Table 2).
TABLE
3. Arterial ( A ) and arteriovenous (A-V) concentrations of amino acids in blood across the forelimb of four normal
subjects aflerprotein (steak)ingestion
Grilled steak (250 g raw weight) was ingested between 0 and 15 min. Values (in pmol/l, or munits/l for insulin) are means &
SEM (four subjects). Results significantly different from the mean of pre-ingestion (basal) measurements (four subjects):
* P < 0.05;*+P < 0.01.
Time alter
protein ingestion
(min) ...
Tau
ASP
Ser + Thr
Glu
Gln
G~Y
Ala
Val
lleu
Leu
TYr
Phe
Orn
LY s
His
Try
Arg
lnsulin
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
A-V
A
Concentrations
Basal
30
241 f 18
-5f4
234 f 48
+3 f 3
221 f 10
-8 f 10
175 f 11
+I6 f 2
558 f 43
-39 f 11
305 f 26
-11 f 3
244 f 12
-39 f 9
204 f 10
-2f2
69 f 3
-8 f 2
119f4
-2f2
52 f 5
-4 f 2
43 f 6
-1 f 6
79 f 2
238 f 21
Of 5
267 f 50
+ 1 f 11
297 f 15.
+ 1 f 11
185 f 8
+23 f 4
590 f 53
-51 f 8
315 f 34
+ 7 + 13
264 f 7
-202 12
222 f 5
-6f8
85f 17
+7 f 4'
143 f 20
+11 f 6
53f4
+3 f 4
36 f 6
-1f6
80 f 3
-2f6
191 f 25
-3 f 10
122 f 35
+5 f 8
I I2 f 14
-2*4
92 f 3
+8f4
Of2
184 f 16
-If2
104 f 34
Of4
107f 18
-3 f 5
7 6 f 16
-6 f 5
5f1
-
60
290 f 19
+4 f 5
244 f 45
-
233 f 23
+ 5 f 17
193 f 10
+24f7
617 f 51.
-36f 7
331 f 21
-5 f 11
321 f 9'
-6&6*
259 f 22.
+2f9
146 f 15..
+34 f 12'
231 f 12"
+45 f 5..
I l f 6.
+6 f 2..
58 f 5
-5f4
93 f 5.
+If6
294 f 16"
+11+4
115 f 32
-8f21
1 1 2 + 20
-2OflI
130 f 4.
+18f3*
14 f 4
90
276 f 11
-8-1. 13
246 f 13
f 3 f 13
-
120
283 f 22
-2 f I
273 f 47
-1Of 12
263 f 16
+7 f 16
243 f 34
+3 f 22
-
-
-
193 f 22.'
+26f2
646 f 54'
-35 f 13
365 f 25.
+24 f 20
353 f 17'
-4f7'
345 f 14.'
+29f7*
167 f 12.'
+19 f 11'
262 f 19'.
+40 f 11.
85 f 7.
+5 f 5
64 f 7
-2f5
106 f 6'.
+14+6
333 f 13'.
+15f9
128 f 18
-123
9 2 f 27
Of4
137 f 13'
+13f4**
203 f 10..
+29f4
630 f 49'
-49 f LO
357 f 15'
- 3 f 15
356 f 21.
-4f4.
371 f 18"
+27f9*
177 f 14'
+24f11'
305 f 37.'
+37 f 12'
95 f 7-If10
7 6 f 5.
+3 f 4
115f3**
+4 f 7
371 f 29''
+21f11
133 f 20'.
+3+6
116f21
-2f6
149 f 14'
+12f4"
14 f 3
190 f 11'
+19fZ
639 f 39.
-48 f 12
344 f 14
-11 f 22
363 f 16'
-12f8
369 f 23..
+11 f I1
162 f 6'
+If6
262 f 15.'
+11 f 8
93 f 8'.
-1f5
69 2 4 .
-2 f 5
113f4"
-5 f 5
353 f 24..
-2f17
127 f 23
-5 f 3
118f24
+7f4
136 f 21.
-4 f 9
-
-
I50
-
180
278 f 20
-
276 f 44
+2+6
-
194 f 12.
+20 f 6
610 f 34
-33 f 15
341 f 17
Of9
322 f 20.
-16 f 7
368 f 39.
+6 f 10
161 f 21.
-1 f 12
257 f 28'
+7 f 13
91 f 12.
+2 f 4'
71 f 4 .
-5 f 6
-
320 f 26'
3f7
11Of 16
-727
117f9
-2f2
132 f 26
-2f2
9f3
Branched-chain amino acid metabolism
Forelimb amino acid exchange
Steak
1
I
>
-
g
523
In the basal state alanine and glutamine
together accounted for more than 50% of those
amino acids with a negative A-V (Tables 1 and
3). After ingestion of steak the negative A-V for
alanine decreased by 90% of the basal value
(Table 3), a directional change shared by most
other amino acids (Table 3). In marked contrast,
the negative A-V for glutamine was unchanged,
so that by 2 h after ingestion of the steak,
glutamine accounted for approximately 75% of
those amino acids with a negative A-V (Fig. 3).
After administration of leucine the A-V for
alanine, and for most other amino acids, was not
significantly altered (Table 1). Glutamine again
did not follow this pattern; its negative A-V
doubled and its blood concentration increased
significantly (Table 1).
-40
-120
-160
I
I
I
200
+40
-30
0 .+30 60 90 120 150180
Time (min)
FIG.3. Effects of steak ingestion (250 g raw weight)
on amino acid exchange across the forelimb. (a) Net
A-V difference of all amino acids. (b) Sum of A-V
values for those amino acids with positive A-V values.
(c) Sum of A-V values for those amino acids with
negative A-V values. Contributions made by
branched-chain amino acid (BCAA) and glutamhe
(Gln) are indicated by the shaded areas (n = 4).
In normal subjects leucine administration was
followed by a large positive A-V for leucine
(Table l), but, as after ingestion of steak, the
negative A-V for BCOA increased only slightly
(Table 2).
The above effects were observed in all subjects.
Eflects of ingested steak and leucine administration on intermediary metabolism
After administration of leucine to the normal
subjects, their mean arterial blood glucose concentration decreased significantly (P < 0.01)
from 5.18 f 0.14 in the basal state to 4.87 f
0.11 and 4-85f 0.11 mmol/l (mean f S E M , ~=
4) at 30 and 60 min respectively. The A-V for
glucose also showed decreases at these times
when compared with the basal values, from
+0.19 f 0.04 to +0.10 f 0.03 (P < 0.05) and
+0.15 f 0.05 mmol/l (not significantly different)
respectively. By contrast, over the 3 h period after
ingestion of the steak, no significant effects were
observed on either the arterial concentration or
the A-V value for glucose (data not shown).
Both arterial lactate and arterial pyruvate
concentrations were elevated (P < 0.01) after
ingestion of steak, from basal values of 0.56 f
0.05 and 0.052 f 0.005 mmol/l respectively to
maximal levels at 90 min of 0.71 f 0.06 and
0.077 f 0.010 mmol/l respectively. At these
times the A-V values for lactate and pyruvate
had changed maximally from -0.10 f 0.04 in
the basal state to -0.06
0.03 mmol/l for
lactate (change not significant) and from -0.006
f 0.002 in the basal state to +0.001 f 0.004
mmol/l (P 0.01) for pyruvate. By contrast,
administered leucine had no effects on either the
arterial or the A-V values for either lactate or
pyruvate (results not shown).
Administration of leucine significantly (P <
0.01) elevated arterial ketone body concentrations from a basal value of 0.27 f 0.07 to a
maximal value at 30 min of 0.44 f 0.08 mmol/l,
5 24
M.Elia and G . Livesey
due mostly to more acetoacetate. This was
associated with a small, though not statistically
significant, rise in the positive A-V value for
ketone bodies. By contrast, ingested steak decreased circulating ketone body concentrations
from a basal arterial valueof0.17 f 0.03 to 0.10
f 0.07 mmol/l at 90 min and this was associated
with a maximal depression in the A-V value for
ketone bodies from a basal value of +0.033 f
0.010to +0.013 f 0.008 mmol/l (P< 0.05).
Contribution of muscle and superficial tissues
to the forelimb arteriovenous direrence
measurements
Since the present studies were undertaken
without using a wrist cuff the arteriovenous
measurements represent exchanges of metabolites
with both the forearm and the hand, and will
therefore have some contribution from both
muscle and superficial tissues. Concurrent
measurements were made of arterial-superficial
forearm vein (A-SV) metabolite concentrations
and the results (not shown) indicated that the
presently reported exchange measurements reflect
mostly muscle metabolism. This is because the
effects of steak ingestion and leucine administration on all the A-SV values were either
similar in direction to the reported A-V values
but smaller in extent or not significantly different
from basal A S V values.
Discussion
BCAA are found in both plasma and
erythrocytes [24, 251. In man we find BCOA to
be located almost entirely in plasma. This differs
from observations on rat blood, showing
association of BCOA with erythrocytes [26, 271,
although the distribution of BCOA in human
blood appears similar to that in bovine blood
[261. Thus in humans variations in packed cell
volume may appreciably affect the whole-blood
concentration of BCOA. Hence, plasma measurements are used to compare circulating BCOA
concentrations in various groups of patients and
whole-blood measurements are used to assess
sequential changes in BCOA concentrations and
A-V differences.
Whole-body BCAA metabolism
The strong correlation between venous plasma
BCAA and BCOA concentrations (Fig. 1)
suggests that these metabolites are closely linked
by an equilibrium reaction catalysed by the
BCAA transaminases, enzymes which are distributed widely in human tissues [281. Only in the
rested and fasted subjects and patients is the
BCAA/BCOA ratio constant. Protein feeding
(Fig. I), leucine infusion (Fig. 2) and exercise [291
each increase the BCAA/BCOA ratio.
It was shown previously [61 that leucine
clearance rates are decreased in starvation,
muscular dystrophy and insulin-dependent
diabetes and increased in cirrhosis. However, the
plasma BCAA/BCOA ratio remained similar in
all these subjects both before (Fig. 1) and after
(Fig. 2) being challenged with leucine. Hence
changes in leucine transport into cells or transamination are unlikely to account for the varied
rates of leucine clearance in these clinical conditions. Regulation by phosphorylation at the
BCOA dehydrogenase complex seems more
likely [301.
Branched-chain amino acid oxidation in limb
tissues
The apparent increase in BCAA uptake by the
forelimb tissues of normal human subjects after
ingestion of steak (as judged by changes in the
A-V values) is associated with only minor
changes in the A-V for BCOA (Table 2). At this
time, either BCAA is poorly metabolized by the
forelimb tissues or any BCOA produced is
preferentially oxidized rather than released. It is
probable that BCAA catabolism does occur
since, as judged by the A-V values (Table 3),
these amino acids contribute to approximately
50% of the amino acids entering the limb tissues
after ingestion of steak (Fig. 3) but only 20% of
muscle [31, 321 and skin (D. A. T. Southgate,
unpublished work) protein, i.e. the BCAA, appear
to enter in large excess of any requirement of
protein synthesis. This conclusion is in agreement with the work of Clugston & Garlick 1331
showing 40% of leucine ingested with a meal in
man is oxidized rather than incorporated into
protein.
In an attempt to further increase the rate of
BCAA catabolism and to increase the release of
BCOA by human forelimb, we challenged our
subjects with a leucine load since similar experiments in the rat showed considerable increases in
BCOA release by hindlimb tissues after leucine
administration 1341. Administration of leucine to
man as in the present study produces a massive
increase in the positive A-V for leucine which, in
part (see below), can be explained by a substantial uptake and catabolism of leucine by the
Branched-chain amino acid metabolism
forelimb, but little change in the negative A-V for
BCOA (Table 2). In this experiment, arterial
BCAA and BCOA concentrations decrease
rapidly towards normal values after 15 min
(Table 2), indicating that the A-V difference
measurements underestimate uptake of leucine
and overestimate BCOA release (and vice versa
at 15 min) when accounting for the effect of the
transit time for nutritive blood flow across the
limb [351.
Evidence for leucine catabolism in the human
forelimb after its administration as in the present
study is largely circumstantial. Protein synthesis
cannot account for a large uptake of leucine and
an accumulation of this amino acid within the
tissue would lead to its catabolism at an increased
rate through mass action 121, more especially as
the pool size for leucine in muscle is small [361.
The increase in the negative A-V for glutamine
subsequent to leucine administration (Table I) is
consistent with this conclusion.
Our results (Table 2) suggest either that the
BCAA, and leucine in particular, are not readily
catabolized in the tissues of the human forelimb
or, more likely, that BCOA arising during their
catabolism are oxidized rather than released into
the circulation for further metabolism in other
tissues. In either case other tissues must contribute to the elevation of the BCOA concentration in the circulation after leucine administration. Our results also cast some doubt
about the source of circulating BCOA during the
basal state in man since the A-V measurements
suggest that basal release of BCOA by the
forelimb tissues is small, whereas the capability
for BCOA removal from the circulation seems
large, as judged by the rapid decrease in their
circulating concentration 15 min after leucine
administration (Table 2). The situation appears
different in the rat where muscle contributes large
amounts of BCOA to the circulation for removal
by the liver [ 51.
Nitrogen metabolism in the limb tissues
BCAA are effective stimulators of both alanine
and glutamine release by rat muscle in uitro (for
references see the Introduction). Our results
would indicate glutamine rather than alanine to
be the major vehicle by which nitrogen leaves
human forelimb tissues in uivo after both a
protein meal and an intravenous leucine load.
These observations are for man previously in the
overnight rested and fasted state and we cannot
extrapolate these findings to other metabolic
conditions. After ingestion of the protein meal we
suggest glutamine contributes up to 75% of the
525
total amino acid or 87% of the total amino acid
nitrogen release by the forelimb tissues and that
glutamine, not alanine, is synthesized de nouo
after the intravenous leucine. The latter finding
agrees with that of Aoki et al. [131, who showed
elevated glutamine release after a leucine meal in
man. However, the theoretical possibility that the
elevated plasma leucine displaces intracellular
glutamine into the circulation cannot yet be ruled
out.
Selection of respiratory fuel
The decrease in the positive A-V for glucose
by the forelimb in our overnight rested and fasted
subjects in response to leucine in the present
study is consistent with Sherwin’s finding 1371,
using radiolabelled glucose, of a decrease in
peripheral glucose utilization in starving human
subjects given leucine. We suggest further preservation of glucose carbon by an inhibition or
inactivation of pyruvate dehydrogenase; this is
suggested by an increase in the ratio for the
negative A-V of ‘glycolytic products’ lactate,
pyruvate and alanine (and possibly glutamine) to
the positive A-V for glucose (see reference 1381
for a detailed support of this argument) and finds
support from experiments in uitro on the
inhibition of pyruvate dehydrogenase flux by
leucine or its metabolites 1391. By contrast, this
ratio is decreased after feeding with protein,
which suggests either decreased formation of
‘glycolytic products’ by diversion of glucose
carbon to glycogen or their increased removal by
oxidation at the pyruvate dehydrogenase complex, or both. An increased oxidation of pyruvate
would be consistent with the associated decrease
in the positive A-V for ketone bodies 1381 and
would imply net loss of glucose carbon for
oxidation in the forelimb tissues.
Acknowledgments
We thank Mrs Rosemary Farrell for automated
amino acid analyses, Dr R. Turner for assays of
insulin, Dr B. Winsley for preparing L-leucine
solutions for infusion, the consultants who
allowed the participation of patients and Dr
D. H. Williamson, Dr P. Lund and Dr R. Smith
for helpful discussions. The authors were supported by Medical Research Council Project
Grants to Dr P. Lund and Dr D. H. Williamson
and Dr R. Smith.
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