Download Determination of Pyruvate Oxidation Rate and Citric Acid Cycle

CLIN. CHEM.
24/2,
200-203(1978)
Determination of Pyruvate Oxidation Rate and Citric Acid Cycle
Activity in Intact Human Leukocytes and Fibroblasts
Hans L. WIllems,1
Veerkamp2
Ted
F. M. de Kort,1
Frans
J. M. Trijbels,1
We measured pyruvate oxidation in Intact leukocytes
and
fibroblasts by measuring 14C02 production. The optimal
pyruvate concentration
appeared to be higher than that
usually applied. Activities remained constant during the
incubation and were proportional to the amount of tissue
protein added. Mean values (± SD) were 2.8 ± 0.9 nmol/h
per 106 cells and 37 ± 14 nmol/h per mg of protein for
leukocytes
and fibroblasts,
respectively,
uvate oxidation;
and 2.1 ± 0.8 nmol/h
for [1-14CJpyr-
per 106 cells and
18 ± 7 nmol/h per mg of protein, respectively,
‘4C] pyruvate oxidation. We compared
oxidation
pyruvate
and 2-oxoglutarate
by intact
cells
for [2-
rates
with those
of
of
isolated mitochondria.
The ratio of 14C02 production vs.
activity of mitochondrial marker enzyme demonstrated
that
the rate of pyruvate
in intact cells, but
oxidation
can adequately
be assayed
that the permeability of the cell membrane is rate-limiting in the oxidation of 2-oxoglutarate. No
significant oxidation of other intermediates
of the citric acid
cycle was found, presumably
owing to a low rate of
transport of these substances
across the cell membrane.
‘
Keyphreases:
pyruvate
oxidation
use of intact
cells to measure
enzyme
activity
transport
across
the cell
membrane
2-oxoglutarate
pediatric
chemistry
.
lactic
acidemia
AddItional
During
the last years there has been increasing
interest
in determining
enzyme
activities
in cultured
fibroblasts
and leukocytes.
Though
enzyme
defects
can
be established
in liver, kidney,
or muscle
biopsy
materials, these studies
are rather
limited.
Frequently
only
fibroblasts
are available
for enzyme
assays.
Because
leukocytes
can easily be obtained,
they are also an important
source for the study of enzyme activity.
We have
preferred
to work with intact
cells, so as to approach
physiological
conditions
insofar
as possible.
Use of intact cells has the advantage
that the intracellular
relationships
are intact.
Moreover,
the cytoplasm
and the
intra-mitochondrial
content
are diluted
on sonication,
which necessitates
addition
of cofactors
to the incuba-
Departments
of Pediatrics,1
and Biochemistry,2
Nijmegen,
Nijmegen,
The Netherlands.
Received
Oct. 11, 1977; accepted
Dec. 1, 1977.
200
CLINICAL
CHEMISTRY,
Vol.
24,
No.
2, 1978
University
of
Leo A. H. Monnens,’
and Jacques
H.
tion medium.
Our procedure
is superior
to most of the
previous
methods
because
deficiencies
of cofactors
may
be established
as well.
Our aim in the present
investigation
was to study the
contribution
of transport
across the cell membrane
in
the assay of pyruvate
oxidation
with [1-14C]pyruvate
and the citric acid cycle activity
with [2-14C]pyruvate
and [1-’4CJ2-oxoglutarate
in intact
leukocytes
and fibroblasts.
Materials
and Methods
Leukocytes
One volume
of 50 g/liter
dextran
solution
(T500;
Pharmacia,
Uppsala,
Sweden)
containing,
per liter, 7
g of NaCl and 15 g of disodium
ethylenediaminetetraacetate)
was added
to five volumes
of blood.
Blood
samples
were obtained
after an overnight
fast from
children
(1-10 years),
all hospitalized
for disorders
not
interfering
with pyruvate
metabolism.
The total leukocyte
count was in the range 5.10#{176}
to 10.10#{176}
per liter.
The differential
leukocyte
count was: 60-70%
neutrophils,
20-30% lymphocytes,
and 2-10% monocytes.
After
30 mm at 4 #{176}C
the supernatant
plasma-containing
leukocytes,
some
erythrocytes,
and
platelets-was
centrifuged
for 8 mm at 300 X g. The supernatant
plasma,
which
contained
most
of the platelets,
was
discarded.
The pellet was suspended
in 1 ml of buffered
saline
(40 mmol/liter
phosphate
buffer,
pH 7.4, containing
0.11 mol of NaC1 per liter).
The remaining
erythrocytes
were removed
by applying
an hypotonic
osmotic
shock by exposure
to distilled
water for 90 s,
after which isotonicity
was restored
(1). After washing
with saline,
the final pellet was suspended
in buffered
saline and the cells were counted
in a Coulter
Counter.
Mitochondria
were prepared
from a concentrated
suspension
of leukocytes
(30.109/liter)
in 0.25 mol/liter
of sucrose.
The cell membrane
was disrupted
with a
French
press at 3.5 X 106 Pa. The cell debris
was removed
by centrifugation
(10 mm at 70 X g) and the
mitochondria
obtained
by centrifugation
for 13 mm at
8000 X g were washed
once with 0.25 mol/liter
of sucrose.
Fibroblasts
tainate
dehydrogenase
(EC 1.4.1.2) activity
were measured
in
cells
and
mitochondria,
both
after
freezing
and
Skin biopsy
material
was cut into small fragments
thawing
three
times.
These
assays
were
performed
acand cultured
in TC-199
medium
(Flow Laboratories,
cording
to
Cooperstein
and
Lazarow
(4)
and
Schmidt
Irvine,
Scotland)
enriched
with, per liter, 200 g of fetal
(5), respectively.
calf serum and 50 g of chicken
embryo
extract,
suppleThe respiratory
control
index of mitochondria
of fimented
with 1 X 10 USP units of penicillin
and 100 mg
broblasts
was
measured
in
simplified
Dow
(6)
medium
of streptomycin.
After trypsinization
for 5 mm at 37 #{176}C
containing
1 mmol of pyruvate
and 1 mmol of malate
(2.5 g of trypsin
per liter; Difco, Detroit,
Mich.;
1:250
per
liter.
ADP
was
added
in
a
final
concentration
of 0.2
pancreas)
the fibroblasts
were washed
with the phosmmol/liter.
phate-buffered
saline. The cells were counted
under the
microscope
in a special
glass counting
chamber
after
Radiolabeled
Materials
dilution
of the cell suspension.
[1-’4C]Pyruvate,
[2-14C]pyruvate,
and [1-’4C]2-oxoFor isolation
of mitochondria,
fibroblasts
were susglutarate
were obtained
from the Radiochemical
Centre,
pended
in a medium
containing,
per liter, 0.27 mol of
Aniersham,
and
stored
at
-20
#{176}C
in
small
aliquots
as the
mannitol,
0.1 mmol
of disodium
ethylenediaminetedry
sodium
salts,
under
nitrogen.
traacetate,
0.5 g of bovine serum albumin,
and 10 mmol
of tris(hydroxymethyl)aminomethane#{149}HC1
(pH 7.3).
Results
The cells (4.109/liter)
were disrupted
at 4 #{176}C
with a
Isolation of Leukocytes
and Fibroblasts
Potter-Elvejhem
homogenizer
and the mitochondria
were isolated
by differential
centrifugation
for 7 mm at
The differential
leukocyte
count remained
unchanged
770 X g and 10 mm at 10 000 X g.
after isolation
in comparison
with that in the peripheral
blood. Recovery
of leukocytes
in the final preparation
Enzyme
Assays
averaged
50-60%;
6% of the original
amount
of platelets
Leukocytes
(5.106
to 10.106) were incubated
in the
was found
in the final leukocyte
suspension,
which
phosphate-buffered
saline
and either
pyruvate
or 2contained
on the average
equal amounts
of erythrocytes
oxoglutarate
(0.5 mmol/liter),
the final volume being 2.1
and leukocytes.
ml. The specific
activity
of the used substrates
was 1.0
After
trypsinization,
2-5% of the fibroblasts
were
non-viable,
as judged
by trypan
blue uptake.
mCi/mol.
Incubation
was done in a shaking
waterbath
at 37 #{176}C
in 20-ml glass scintillation
vials, sealed
with
Isolation of Mitochondria
rubber
stoppers,
containing
two small tubes, one fitting
inside the other (2). The reaction
was stopped
after 60
Leukocytes
are relatively
resistant
to the usual homm by injecting
0.3 ml of 3 mol/liter
of perchloric
acid
mogenization
procedures
(7). The best disruption
of
and the ‘4C02 produced
was trapped
in Hyamine
hycells was obtained
by a more vigorous
method:
use of a
French
press. The respiratory
control
index of the midroxide
[p -(diisobutyl-cresoxyethoxyethyl)dimethylbenzylammonium
hydroxide]
during further
incubation
tochondria
of leukocytes
could not be established
befor 30 mm at 37 #{176}C.
The trapped
‘4C02 was measured
cause of the low oxidative
capacity.
after adding
10 ml of scintillation
fluid (4 g of Omnifluor
Fibroblasts
could be well homogenized
with a Potper liter of toluene)
in a liquid scintillation
counter.
For
ter-Elvejhem
homogenizer
in isotonic
medium.
The
respiratory
control
index of the isolated
mitochondria
the determination
of the blank
values
the cells were
was 4.0 with pyruvate/malate
as substrates.
omitted.
Fibroblasts
(5.10
to 1.106)
were incubated
under
identical
conditions
as described
for the substrate
concentrations
oxoglutarate
(0.2 mmol/liter).
The
ml. Protein
was assayed
according
for leukocytes
except
of pyruvate
and 2final volume
was 1.5
to Lowry et al. (3).
Mitochondria
Pyruvate
oxidation
by mitochondria
isolated
from
fibroblasts
and leukocytes
was measured
at 37 #{176}C
in an
incubation
mixture
containing,
per liter, 50 mmol of
potassium
phosphate
buffer
(pH 7.4), 0.5 mmol of disodium
ethylenediaminetetraacetate,
2 mmol of ADP,
75 mmol of KC1, 10 mmol of MgCl2, 1 mmol of the substrate
(0.5 mCi/mol),
and 0.2-0.4
mg of mitochondrial
protein.
The final volume
was 0.5 ml. Incubation
time
was 60 mm.
Leukocytes
and fibroblasts
yielded
about 4 and 40 mg
of mitochondrial
protein
per 10#{176}
cells, respectively.
The
assay
procedure
Cytochrome
was as described
for
c oxidase
(EC 1.9.3.1)
intact
activity
cells.
and
glu-
Enzyme
Assays
14CO2 production
from [1-14C]pyruvate
was lower
without
added
cells than that in the presence
of boiled
cells. Nearly identical
blank values were obtained
either
in the absence
of cells or after inhibition
of pyruvate
oxidation
with arsenite
(4.4 mmol/liter).
The presence
of boiled cells appeared
to give an incorrect
blank
value
by a higher
nonenzymatic
decarboxylation
of [1-’4C]
pyruvate
(8).
The results
(Figure
1) indicate
that the reaction
rate
of the oxidation
of [1-14C]pyruvate
and [2-’4C]pyruvate
by fibroblasts
remained
constant
during
60 mm of incubation,
and also was proportional
to the amount
of
cellular
protein.
Similar
results
were obtained
for lenkocytes.
Table
1 shows control
values for the pyruvate
oxidation
rate in leukocytes
and fibroblast.s.
The CV for
the assay amounts
to 7%, both for leukocytes
and Iibroblasts.
The Lineweaver-Burk
plots with [1-’C]pyruvate
as a substrate
CLINICAL
revealed
CHEMISTRY.
an apparent
Vol.
24, No.
Km
2, 1978
value
201
-
nrnol
Table
1. Pyruvate
Oxidation
In Human
Leukocytes
and Cultured
Flbroblastsa
I 1-4CJPyruvat.
Leukocytes
I mg
protein
nmol/
h
#{149}-.!1-#{176}CJpyruvote
12-”CJ pyruiat.
100
80
[2-4CJPyruvate
b
2.8 ± 0.9
range 1.4-4.1
60
2.1 ± 0.8
range 1.1-3.5
(n = 11)
(n
12)
=
40
Fibroblasts’
37 ± 14
18 ± 7
range 19-6 1 (n = 10)
range 12-30 (n = 5)
a
Values
are
given
20
± SD.
as the mean
15
106 Ieukocytes.
nmol/h per mg of protein.
b nmol/h
per
in
leukocytes
of 0.14 mmol/liter
of citric
and
fibroblasts
and
acid
cycle
with
its
for fibroblasts
activity
of
in intact
intermediates
2. Pyruvate
intact culttred
rates of [1-14C]pyruvate
fibroblasts
incubated
dehydrogenase
cells
in leukocytes
c oxidase
2-Oxoglutarate
dehydrogenase
c oxidase
Cytochrome
was too low to be used
202
as a reliable
Dehydrogenase
and Leukocytes.
Activity
in Intact
Cells
and
Leukocytes
MitOchOndria
Intact
(nmollh
218
5
77
1242
6930
3060
11 820
Cells
MitochOndria
per mg of protein)
2.9
16.6
0.2
-
492
(X
10.7
-
1270
1O)
30.6
31.5
4.0
11.1
12.4
18.4
5.9
13.0
1.6
6.5
0.5
8.4
dehydrogenase
dehydrogenase
2-Oxoglutarate
Glutamate
of time
concen-
dehydrogenase
Cytochrome
Glutamate
periods
enzyme
Several
clinical
disorders
are associated
with
increased
concentrations
of lactate
or pyruvate
(or both)
in blood or cerebrospinal
fluid (or both).
The mean
clinical
features
of patients
suffering
from these disorders are mental
retardation,
muscular
hypotonia,
and
convulsions
(1). Measuring
pyruvate
oxidation
rate in
leukocytes
and fibroblasts
can be helpful
in the diagnosis of children
suffering
from lactic acidemia.
Leukocytes
and fibroblasts
contain
the enzymatic
equipment
for pyruvate
oxidation
(9-11),
such as pyruvate dehydrogenase
(1), the citric acid cycle (12),
and
the electron
transport
system
(13-15).
The oxidation
rates of [1-’4C]pyruvate
and [2-’4C]pyruvate
we found for intact leukocytes
and fibroblasts
(Table
1) much exceed
those previously
published
(1,
16, 17), perhaps
because
of the higher
substrate
concentration
we used. In other investigations
(1, 16, 17)
pyruvate
concentration
was below the Km value.
Pyruvate
is oxidized
in fibroblasts
more rapidly
than
Ratio
Pyruvate
and [2-14C]pyruvate
for various
Discussion
38
dehydrogenase
Cytochrome
c oxidase
Glutamate dehydrogenase
dehydrogenase
dehydrogenase
CLINICAL
CHEMISTRY,
Vol.
24,
No. 2, 1978
0.5
protein
marker.
Activity
Pyruvate
0.4
trations (incubation time, 1 h)
Flbroblasts
2-Oxoglutarate
03
mg
(using 1.0 mg of protein) and with increasing
activity
Dehydrogenase
and 2-Oxoglutarate
Mltochondria
of Flbroblasts
Intact
Pyruvate
0.2
as
substrates
was almost
impossible.
Citric acid cycle activity could be only demonstrated
by using [2-14C]pyruvate as substrate
(Table 1). No measurable
activity
was
found
with [U-14C]malate,
[1,5-’4C]and [6-”C]citrate,
[1,4-’4C]succinate,
or [5-’4C]2-oxoglutarate
as substrates.
The oxidation
rate with [1-’4C]2-oxoglutarate
and [1-14C]glutamate
as substrates
was low in intact
cells.
Table
2 compares
the oxidation
rates of [1-’4C]pyruvate
and [1-’4CJ2-oxoglutarate
by intact
leukocytes
and fibroblasts
and by their mitochondria.
Cytochrome
c oxidase
and glutamate
dehydrogenase
were measured
as mitochondrial
markers.
The ratio of pyruvate
dehydrogenase
activity
to activity
of mitochondrial
markers
is in the same order of magnitude
for intact
cells and mitochondria.
The discrepancy
of the ratio for
pyruvate
dehydrogenase
to glutamate
dehydrogenase
between
intact cells and mitochondria
might be due to
leakage
of glutamate
dehydrogenase
out of the mitochondrial
matrix.
The ratio for 2-oxoglutarate
dehydrogenase
activity
was, however,
much higher in mitochondria
than
in intact
cells. Cytochrome
c oxidase
Table
0.1
60
mm
Fig. 1. Oxidation
for leukocytes
0.11 mmol/liter.
Determination
45
30
in leukocytes.
The oxidative
pathway
in leukocytes
is
of minor
importance
(18).
The generation
of ATP
quantitatively
depends
less on oxidative
phosphorylation than on glycolysis
(7).
The approximately
equal
‘4C02
production
from
[1-’4C]pyruvate
and [2-’4C]pyruvate
with intact
leukocytes
and fibroblasts
(Table
1) contrasts
with the
results presented
by Cederbaum
et al. (16) and with the
observations
on rat adipose
tissue (19). Our data suggest
that in leukocytes
and fibroblasts
acetyl-CoA
produced
from pyruvate
is almost completely
oxidized
in the citric
acid cycle under
the conditions
we used.
No reliable
measurements
of citric acid cycle activity
could be performed
with intermediates
of the citric acid
cycle as substrates
in intact leukocytes
and fibroblasts.
This might be due to a low activity
of the citric acid cycle
or to the limited
transport
of these substrates
across the
cell membrane
or the mitochondrial
membrane.
The
experiments
with [2-’4C]pyruvate
as substrate
show
that in vitro measurement
of the overall citric acid cycle
activity
is feasible.
Moreover,
earlier
studies
(9, 12)
demonstrated
a definite
citric acid cycle activity
in both
types of cells. The low rate of transport
of 2-oxoglutarate
across the cell membrane
will cause a difference
in its
rate of oxidation
between
intact
cells and mitochondna.
We conclude
that pyruvate
oxidation
can be reliably
measured
in intact
leukocytes
and fibroblasts
with use
of [1-14C]pyruvate.
Concomitant
assay of ‘4C02 production
from [2-’4C]pyruvate
can provide
an indication
for the total activity
of the citric acid cycle, but the individual
steps of this cycle can only be determined
by
2. Fox, R. M., A simple incubation
Biochem.
41,578
(1971).
using
carbohydrate
in a patient
with familial
intermittent
lactic acidosis
and pyruvate
dehydrogenase
deficiency.
Pediat.
Res.
10, 713
(1976).
17. Falk, R. E., Cederbaum,
S.D.,
Blass,
J. P., et a!., Effects
of a ketogenic
diet in two brothers
with pyruvate
dehydrogenase
deficiency.
Pediatrics
58,713
(1976).
mitochondria
or
disrupted
cells.
Prolonged
trypsinization
of fibroblasts
may increase
the cellular
uptake
of di- and tricarboxylic
acids (12)
but may disturb cellular
metabolism.
We suggest
that use of intact
leukocytes,
and especially
fibroblasts,
may be appropriate
to detect
defects
in pyruvate
oxidation
clinical-
3.
193,
265
method
189,665
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matischen
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Uhlendorf,
B. W., A defect
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Anal. Biochem.
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Function
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A. E., Leucocyte
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mite-
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K. G., Radiochernical
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drate
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Hohnadel,
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D. S., and Horn, E., An inacid cycle in a patient
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Vol. 24, No. 2, 1978
203