A Gas Chromatographic
Determination
of Lactic
John
A method
tography
Savory*
for the determination
is described.
Lactic
and
Method
for the
Acid in Blood
Alex
Kaplan
of lactic acid concentration
in blood by gas chromaacid in a protein-free
filtrate
is oxidized
by ceric
sulfate
to acetaldehyde.
The latter
compound
is introduced
into
tographic
column
by vapor phase injection,
and is quantitatively
3 mm.
The
method
is specific,
precise,
and
interfere
with the determination
of lactic
A method for preserving
blood samples
concentration
ACTIC
present
ACID
in
l5
blood
(1)
attention
of
been
reported
quada
(2).
Methods
the end product
at a concentration
in
1961
practicing
since
for
that
by compounds
that
may
the
physicians.
time
and
glycolysis
and is normally
mM
(4-12
mg./100
ml.).
concept
of
Many
cases
are
included
lactic
acid
in
lactic
phenazirie
involve
either
is measured
formazan
biologic
either
directly
color
produced
methosulfate
oxidation
of
colorimetrically
method
introduced
chromatography
filtrate
of blood
recently
for measuring
was
mixed
by
the
with
acidosis
to
of lactic
acidosis
in a review
by
by
by
involve
methods
and
(3-7),
NAD+
spectrophotometry
the
interaction
and a tetrazolium
dye.
lactic
acid
to acetaldehyde,
The
(7-9)
(JO-li).
or
titrimetrically
the
have
Tran-
materials
or chemical
oxidation.
In the enzymatic
to pyruvate
by lactate
dehydrogenase
NAI)H
by the
NADH
with
cal methods
of anaerobic
of 0.4-1.4
brought
determining
enzymatic
is oxidized
the resulting
or indirectly
analyzed
affected
acid by colorimetric
or enzymatic
methods.
up to 24 hr. without
change in lactic acid
is also described.
Huckabee
either
lactate
not
the gas chromameasured
within
of
chemiwhich
is
Hoffman
et ci.
(12)
employed
acetaldehyde.
A trichloroacetic
a large
excess
of periodic
acid,
A
gas
acid
and
From
the University
hospital
aiid
1)epartment
of Bioehinistry,
University
of Washington,
Seattle,
Wash.
98U)5.
The
authors
thank
Dr.
Loring
B. Rowell
and
Mrs.
Fusako
Kusunii,
who
ptrfornieil
tlti
analyses
of lactic
acid
concentration
by the Barker-Summerson
method.
Received
for publication
1)ee.
10, 1q65;
accepted
for publication
Feb.
17, 1966.
*postdoctoral
trainee
supported
by Training
Grant
3T1
GM
776-02,
in Clinical
Chemistry,
from
the National
Institute
of General
Medical
Sciences,
U. S. Public
Health
Service.
Present
address:
University
of
Florida,
College
of
Medicine,
559
Gainesville,
FIn.
560
SAVORY
1
of
graph.
the
mixture
Water
ing
a high
tion
took
was
column
acid
eliminated
in
of the
carrier
wait
was
sugars
retention
to
are
adclitiollal
step
of
A rapid
and
acid
the
l)laCe
and
in
was
the
method
a gas
and
at
which
developed
by
acetaldehyde
as
of
of the
another
changing
this
and a. 15(3) The
temperature,
the latter
has
with the analysis.
tiherefore,
required,
to
method
avoid
the
onto
a vapor
ti-ichioroacetic
column
sample.
100#{176}.At
would
ci.
The
nonvolatile
continual
fornialdehyde;
and interfered
oxidizing
the
ct
(1)
many
The
chironiatographic
blood
iii
was
injecting
took
through
determination
and
was
flow had to be increased
gas
the stability
injection
of
leavoxida-
Hoffman
amount
each
cai-rier
chromato-
problems:
elimination.
to fol-mic
acid
to acetaldehyde
acid
lactic
this
i-ate upset
before
sugars.
made
foi-
method
with
the
up
periodic
oxidized
tune
similar
these
interfering
A search
was
uremeiit
speed
of
acid
large
column
gas
swept
method
periodic
a
Chemistry
of the column,
acid.
A rapid
technical
A relatively
the
gas flow
necessary
with
oxidation
with
(2)
of
was
the
following
Consequently,
order
formed
hands,
the
into
slowly.
sixfold
mm.
blood.
injected
column
hot injection
port
acid and periodic
our
have
Clinical
the
acetaldehvde
Ill
contaminated
of
was
the
to
became
components
An
and
found
into
in the
of lactic
measured.
and
(12)
injected
evaporated
concentration
place,
column
was
& KAPLAN
the
lactic
the
the
for
above
acid
gas
remove
nieas-
difficulties.
in
a test
tube
chromatographic
column.
Lactic
dehyde
method.
acid
by
m
a protein-free
ceric
sulfate,
(‘oiitaniination
by injecting
tion utilized
of the
the acetaldehyde
a technic
described
determinatioii
dation
mixture
of blood
alcohol.
was
added
to
copper
sulfate.
The
sure
which
liberated
allowed
the
Only
volatile
was
column
injection
lower
sensitivity.
water
peak
Our
eliminated
was
obtained
the
need
oxidized
rapidly
Long
column
be injected
phase
hydrate
solution.
every
inect.ion
from
injecting
a solution
of the
Natelson
and
for
the
perforated
of low
ilelease
avoided
of
injecfor the
also
The
heads.
sensitivity
since
the acetalConventional
oiiwith
concomitant
avoided
directly
(13)
vapor
presof a clamp
gas chromatograph.
and
contamination
3 mm.
Stellate
glass
was
the ceric
sulfate
oxi150 mg. of anhydrous
by this type
of injection
of solution
was
analyzed.
to 1 or 2 jJ. of solution
vapor
acetal-
titrimetric
state.
rllhis method
and
Stellate
(i.)
An aliquot
from
a tube
containing
a stable
from
to
in his
(ii)
chromatographic
in the vapor
by Natelson
could
increased
in 50 l.
is limited
The
modification
gas
by
vapor
to be swept
into the
was
applied
to the column
1 sample
analysis
contained
was
used
water
formed
the acetaldehyde
acetaldehyde
material
eliminated;
of tile
dehvde
filtrate
a reagent
onto
injection
The
use
the
the
large
column.
chamnbem-
of a Turbo
a
Vol.
12,
No.
9,
1966
miXeI’
(Technilab
hydrate
formed
powder
being
LACTIC
ACID
iiistrunients)
broke
time dehydration
ill
swept
IN
ilito
tile
up
the
step.
gas
561
BLOOD
cluiiips
There
ol
was
clironiatographic
Material
copper
no
siilhttc
1)i’oblelfl
of
fine
column.
and Methods
Reagents
1.
to
Perehioric
100
ml.
2.
acid
with
Gene
7%
(v/c)
distilled
sulfate
30 gni.
of ceric
Allow
tIme solution
l)ilute
10 ml.
of 70%
perchioric
acid
water.
30%
(w/c)
sulfate
and
to
Add
heat
stand
100
at 70#{176}
until
at
room
nil,
of
iN
a clear
sulfuric
solution
temperature
for
acid
to
is obtained.
12 1w.
and
then
filter.
Litli ium
3.
lactate
mamt-Le(idon
store(l
Lactic
lithium
acid
lactate
This
5.
Lactic
tion
standard,
100 nil.
is stable
acid
1 week
of tile
l)L-lithium
overnight
lactate
in a vacuum
(Hart-
desiccator
and
bottle.
stock
in
solution
commercial
is dried
at 4#{176}
in a dark
4.
for
I 5urifled
Company)
of distilled
for
working
when
stored
stock
standard
20.0
mM
water
several
store
and
191.8
ill
the
mg.
(lark
of
at
4#{176}.
months.
standard,
0-5
at 4#{176}
in the
dark.
with
l)issolve
mM
This
Prepare
distilled
solution
by
is stable
appropriate
dilu-
watem’.
Apparatus
Gas
chromatograph
A
Model
5000
gas
chroinatograph
Coleman
Company)
was used with a hydrogen
and a trn-shaped
column,
6 ft. X ?g iii., packed
silicone
Engineering
guni
rUl)ber
SE-30
Laboratories,
tained
at 75#{176}
and
120 ml./mniii.
The
on 70/80
Inc.).
The
time carrier
temperature
Dehydration-injector
means
of hypodermic
mesh
Aitakrom
AB
column
temperature
gas
(nitrogen)
of the detector
system
needles
flow
bath
A dehydration
plastic
tul)ilIg
aiid
(Barber-
flame
ionization
detector
with
25% by weight
GP-88
(Analytical
was
main-
rate
was adjusted
was 250#{176}.
to
tube
connected
to a nitrogen
tank
1w
and
gas chromatographic
acetaldehyde
vapor
injector
12-
system
X 75-mm.
powder
and
column
served!
as an inJector
system
for sweeping
into the column.
A schienmatic
representation
of this
is shown
in Fig.
1. The
dehy(lration
chamber
was
a
test tube containing
150 fig,
of alillydrous
cOppem
sulfate
was
sealed
by
a punctui’e
stopper;
Becton,
1)ickinson
(No.
20, 1.5 in. and No. 21,
with hose end were
inserted
The
hose
ends
were
connected
type
and
Company).
1 iii.)
through
connected
the
to
h-glass
rul)l)er
stopper
Two
to
rubber
a male
stopper
fittings
(Vacutainer
hypodermic
needles
Luer
connection
of the chamber.
(Technicon
Instru-
SAVORY
562
ments
Corporation).
plastic
tubing
All
& KAPLAN
connections
of 0.045-in.
Clinical
shown
iii
Fig.
i were
ml.
of 7%
(v/v)
5 or
10 mum,
Chemistry
made
with
I.D.
Procedure
Protein
Precipitation
Pipet
and
1.0 ml.
of whole
blood
for
30 sec.
mmlix vigorously
until
a clear
supernatant
into
Let
layer
1.0
stand
for
perchioric
and
acid
centrifuge
is obtained.
Fig.
1. Schematic
resentation
tioii-injector
dehy
chamber;
CHRO,- VA TO
ends;
fittings;
GRAPH
dehydrasystem.
indicates
GAS
rep-
of
B
C
A
d ration
and
B’,
hose
and
C’, h-glass
D and
D’, hypo-
dermic
needles;
X,
tic tubes;
Y, plastic
plastube
by-pass.
Oxidation
Transfer
1.0 ml.
o.r
of
Cool
to room
ml.
Incubate
Extraction
of supernatant
30%
ceric
the tube
for
and
fluid
sulfate
10 mm.
temperature
to a 12-
and
seal
tightly
at 37#{176}
in order
and
X 75-mm.
test
with
a
to complete
rubber
tile
tube.
Add
stopper.
oxidation.
centrifuge.
Injection
A schematic
representation
Anhydrous
copper
sulfate
of time injector
powder
(approximately
system
is showii
in Fig.
150 mg.)
is dispensed
1.
into a 12- X 75-mm.
test tube
(Fig.
1, A) which
is sealed
with a puncture
type
rubber
stopper.
Several
of these
tubes
are
prepared
in advance
of a run since
the copper
sulfate
is stable
indefinitely.
A 50-l.
sample
of the
100-l.
supernatant
Hamilton
fluid from
syringe
stopper
onto
the
tapped
firmly
on
copper
the
bench
the oxidation
(Hamilton
sulfate.
and
The
vibrated
procedure
Conipan,
needle
on
is
is injected
from
a
Inc.)
through
the
removed
a Turbo
and
mixer
the
for
tube
30
sec.
Vol.
12,
No.
9,
1966
Plastic
tubes
dermic
Another
needles
plastic
LACTIC
(Fig.
1, X)
are
are inserted
tube
(Fig.
ACID
IN
clamped
563
BLOOD
with
a pinchcock
and
through
the rubber
stopper
1, Y) serves
as a by-pass
for
the
2 hypo-
of the chamber.
the nitrogen
flow
from
the taiik
to the gas chromatographic
colummi
when
the tubes
are
clamped
at X. The gas flow caim be directed
through
the chamber
to the
column
by removing
the clamp
at X and c1alfl)fl1g
the by-pass
tube at I’.
Thus,
the
nitrogen
sweeps
column.
sociateci
In
our
with
time volatile
experience,
this
type
material
tile
of
injection
hydrogen
flame
a few seconds
of the flame
does not appeal’
from
change
in
almost
tile
(A)
chamber
carrier
gas
always
extinguishes
after
the injection.
to affect
tile stability
into
pressure
Immediate
of time gas
as-
the
relighting
chromato-
graph.
The
acetaldehyde
narrow
Standard
peak
from
on
the
the
oxidation
gas
of
A standard
curve
was
solutions,
mg./100
in Fig.
varied
prepared
ranging
ml.)
by treating
the
For
5.0 mM)
of lactic
acid
iii the
origin.
routine
same
were
Values
of unknowns
lactic
acid determinations,
included
It is possible
concentrations
recording
by
system.
of
from
0.2
manner
as
produced
acid, with
were
and
with
a gas
chromatograph
merely
changing
the
Time
distance
A peak
of
pen
travel
Acid
Oxidation
obtained
cover
is
lactate
mM
blood.
As
in the
the curve
(1.8shown
reaction
passing
standard
(1.0 and
by Ceric
at
a wide
(sensitivity)
inversely
the height
at an attenuation
of 100. For
all peak
heights
reported
in this investigation
to a common
attenuation.
of Lactic
height
to
attenuation
attenuation.
an
range
of
of
the
proportional
attenuation
the
of
300
lactic
temperatures
dehyde
14-17%
acid
solutions,
ranging
1.0
from
concon-
for
mm.
Sulfate
and
5.0
was
achieved
at 37#{176};the peak
higher
at 37#{176}
than
at 60#{176}.
The
at 25#{176},
requiring
temnperatum’e
30 mm.
to attain
of 37#{176}
was
mM,
25#{176}
to 60#{176}.
The
selected
the
heights
oxidation
maximum
because
were
oxidized
highest
yield
for both
proceeded
peak
it provided
is
sake
of
have
been
of Temperature
Two
tion
a
Discussion
one-third
venience,
verted
Extent
lithium
to 20.0
read
from
the
only 2 standards
to the
at
gives
in time m’ui.
Results
Effect
acid
recorder.
a series
in concentration
2, time peak
heights
of time acetaldehyde
directly
as the concentration
of lactic
through
curve.
The
lactic
chi-oniatographic
Curve
standard
180
produced
symmetrical
10
of
solutions
more
height.
were
slowly
An
the
acetal-
oxida-
optimum
564
SAVORY
& KAPLAN
Clinical
Chemistry
yield
of acetaldehyde
in the shom’test
time.
The lower
yields
of acetaldehyde
obtamed
at temperatures
of 50-60
iiiay be ascribed
to further
oxidlatioli
of acetaldehyde
to acetic
acid.
This
loss
of acetaldehyde
was
(lenlonsi
rated
at 60
was
Effect
by a 50%
extended
decrease
froni
peak
iii
10 mliii.
height
to 30
wliemi
the
oxidation
time
miii.
of Time
The
1 .0 and
5.0 mM
staiidards
wei’e
I)eak
height
achieved
a maximum
least
3() nun.
All results
reported
oxidation
temperature
of 37#{176}
for
Completeness
Ceric
oxidized
after
5
iii
this
at 37
mm.
and
The
.
was
were
study
acetaldehyde
constant
obtained
for
at
at
an
10 miii.
of Oxidation
sulfate
was
verting
lactic
sulfate
concentration
oxidation
used
aci(l
were
to
by
Long
similar
less
(ii)
acetaldehyde.
severe;
as
The
to
that
37#{176}
for
10
an
oxidizing
present
of
mm.
agent
method
Long,
but
was
used
fom’
used
the
cOIl-
a cem’ic
conditions
instead
of
of 60#{176}
for
7/
2c
iO
a
4
2
1,0
20
peak
2. Standard
is
plotted
attenuation
J000
from
curve
against
of
30
1 00.
to
100
for
determination
concentration
Actual
in.
attenuations
of
of
6.0
Aced conc.nt-sztion
Lactic
Fig.
-5.o
4.0
3.0
lactic
acid,
lactic
acid.
All
were:
100
from
(1)
Variation
peak
0 to
3L0
in
height
heights
have
10
300
in,;
of
been
from
acetaldehyde
calculated
10
to
to
30
in,;
Vol.
12,
No.
9,
1966
LACTIC
ACID
IN
30 nun.
The question
arose,
thei’efom-e,
lactic
acid
was
complete
after
heating
proposition,
equimolar
prepai’ed.
ard solution
aration
in a cold
of acetaldehyde
adding
0.5 ml.
up to
made,
1000
and
ahqiiots
of
subjected
to
room
at
sulfate
heights
correspondmg
(14).
of
heights
Table
1.
the
from
PEAK
the
mm.
whether
10
and
temperature
IIEIGII’rs
also
acetaldehyde
acid
tile
lactic
FROM
standard
acid
Peak
by
to
(mM)
solutions
AeuI)
chroma-
and
ranged
AND
from
FROM
height
(in.)
Lactic
acid
(
notivt ion
3,3
110
6,6
6.6
100
4.60
18.0
17.0
9.20
375
the
essentially
adequate
of blood
complete
acetaldehyde
lactic
acid
be oxidized
in the
from
of lactic
94.5
93.5
corresponding
increased
oxidation
tactic
aretoldehyde
to
3.0
of
110%
of
Conversion
1.84
93.5%
time
S’CANDARD
0.92
celltratiofl
that
the
of
in Table
SOLUTIONS
sot ution
Acetatdehyde
gas
summarized
acid
Concentration
gravity
prepared
by
and making
solutioiis
are
LA(”l’lC
OxIDIzEl)
were
a standthe prep-
specific
was
water
alialyzedi
solutions
ACETALDF.HYDE
acid
made
out
the
of
this
dilutions
of this
solution
were
w-ere measured
after
injecting
The
lactic
acid
solutions
were
and
lactic
oxidation
To test
lactic
A 9.20-mM
solution
to 800 miii. of distilled
oxidation
equimolar
peak
to
37#{176}
for
acetaldehyde
1#{176},
at which
is 0.8090
of acetaldehvde
ceric
as
at
has a boiling
point
of 21#{176},
which
to prepare.
It was necessary
to carry
ml. with
water.
Approj)riate
the acetaldehyde
peak
heights
into
the gas
chroniatograpii.
tography.
Time peak
1. The
solutions
Acetaldehvde
difficult
565
BLOOD
acetaldehyde
0.920
acid
peak
mM
to 9.20
to acetaldehyde
mM.
Ileights
the
con-
This
demonstrates
by eerie
sulfate
as
was
in
10 mill.
at
37#{176}.The
difficulty
in preparing
an
standard
makes
it essential
for time measurement
levels,
to use a lithium
lactate
standard
which
imiust
same
manner
of the
proposed
as
the
blood
filtrates.
Precision
The
precision
method
analyses
of 14 blood
samples.
Separate
duplicates.
The
range
of concentration
with
a mean
of 1.38 mM.
The
standard
mM.
S.D.
=
was
investigated
by
duplicate
filtrates
were
made
for
time
varied
fi’om
0.55 to 3.80 muM,
deviation
(S.D.)
was
±
0.039
566
SAVORY
where
d stands
for
the
& KAPLAN
differences
Clinical
between
duplicates,
and
Chemistry
N
is
the
total
number
of determinations.
The coefficient
of variation
(S.D.
X 100)
Meais
for the method
with
those
of
was 2.8%.
Tlmis
existing
methods
degree
for
of precision
the determination
compares
of
favorably
blood
lactic
acid.
Recovery
Tests
The
lactic
tained
(10.0
pJ.
acid
sodium
mg./100
of
concentration
fluoride
ml.).
To
a 22.2-mM
enriched
perchloric
determined.
The recovery
2). The
final
varied
Comparison
with
lactic
by
agreed
between
the
for
The
time
within
with
to
solution
of
2
ml.
200
of
acid
(yl.)
this
lactic-acid-
volume
of 7%
the filtrate
was
97 to 104%
(Table
in this
series
of
from
lactic
11
chromatographic
levels
acid
blood
in 10 of the
samples
and
ranged
in 1 l)lood
copper
whole
in
equal
acid
iii
mM.
acid
methods
2.
mg./100
lactic
an
varied
5.33
of
and
RECOVERY
for
from
was
was
measured
Barker-Summerson
0.5
to
12.0
11 comparisons;
sample
the
mM.
the
The
2
difference
10%.
salts
OF
ACID
ml.
added
blood
determination
calcium
LACTIC
Lactic
of
proteins
blood
con-
Substances
Table
Volume
acid
the
which
was found
to be 1.11 mII
blood
was added
50-50()
adding
of lactic
gas
1.4%
2 methods
colorimetric
treatment
lactic
of
1.61.
lactic
Interfering
The
by
concentration
The
methods
solution.
this
blood,
Methods
acid
methods.
Tests
from
Other
simultaneously
(8)
acid
precipitated
the concentration
of the added
concentration
experiments
The
were
and
of whole
to inhibit
glycolysis,
2.0-rn!.
portions
of
lactic
blood
acid,
of a sample
acid
enriched
to
ADDED
content
of lactic
remove
TO
acid
a large
require
number
BnOoo
FRESH
of
blood
to
Recovered
Added
Found
(mg.)
(nig.)
mg.
0
0.000
0.200
50
0.100
0.297
0.000
0.097
97
100
0.200
0.400
0.200
100
200
0.400
0M15
0.415
104
250
0.500
0.720
(1520
104
500
1.000
L200
1.000
100
of
Vol.
12, No.
9,
compounds,
analysis
1966
including
Also,
(9).
sodium
LACTIC
the
iodoacetate)
Propylene
graphic
known
to
acid
or
amid
method
interfere
those
substances
upon
oxidation
Table
3, solutions
3.
the
determination
seriously
which
interfere
FROM
et
sulfate
compounds
VARIOUS
COMPoUNDs
be
Acetoacetic
p eak
CONCENTRATIONS
acid
of
Ascorbic
acid
peak
acid
200
per
-
0
-
0
-
0
-
J)ihydroxyacetone
0
-
2.0
0
-
Glyceraidehyde
0
-
Glycerol
0
-
acid
fl-Hydroxybutyric
acid
Malic
3.3
-
0
-
0
-
Methionine
Propionaldehyde
Propylene
8.0
glycol
10
3.3
Pyruvie
acid
of 200
mg./1.00
4
0
ml.,
treated
with
-
eerie
sulfate,
and
analyzed
chromatograph.
The
acetaldehyde
peak
heights
were
that
of a 200 mg./100
ml. solution
of lactic
acid
(Table
which
is normally
present
in blood
gave
a measurable
peak.
Propionaldehyde
as high
to produce
butyric
4, and
as
tention
zero
gave
produced
acetaldehyde
acid,
and
ethanol,
2%
of that
derived
detected
acetic
that
small
on
the
time
acid,
of 3
same
mm.
$-hydroxybutyric
interference.
rise
by
to an
lactic
peaks
whose
fromn
peak
The
only
propylene
on
the
gas
compared
with
3). No substance
acetaldehyde
that
other
glycol,
was
10%
compounds
-hydroxy-
respective
lactic
acid.
peak
heights
were
only
Ethianol
per
se could
as
but
to 1
acid
acetaldehyde
acid.
were
chiromatograni
compared
ml.
4
0
acid
lactic
2
Glucose
cr-Hydroxybutyric
peak
to
mg.
100
ML.
(%)
in.)
Acro1ein
Ethanol
in
Mo./100
height
from
0
Acetone
acetal-
shown
200
OF
height
100
(Attenuation.
lactic
to
As
height
Acet&de.hyde
of
at a concentration
Ratio
Compound
acid.
chromatocompounds
tested.
prel)am-ed
AT
gas
converted
were
were
the
(12).
Many
determination
possibly
eerie
the
and
of lactic
in
al.
the
for
might
with
wimich
interfere
with
(sodium
fluoride
enzymatic
ethanol
of these
INTERFERENCE
561
BLOOD
proposed
by Hoffman
in other
methods
dehyde
Table
IN
glucose
and
pyruvate,
effective
preservatives
inhibit
glycol
ACID
and
acetaldehyde,
mm.
for
numerous
thie
it iiad
later.
Acetone,
other
compounds
4,
be
a i’eacetohad
568
SAVORY
Preservation
of Blood
(Jlycolysis
rapidly
is
necessary,
glycolysis
lecting
the
increases
to
The
a chemistry
-as
made
colysis
8 hr.
or
per
aci(l
of
This
plus
preserve
time proteins
is
technician
be
for
a suitable
ionger.
Bueding
4.
acid
only
when
standard
of
glucose,
En’F.cT
added
OF
FlUORIDE
potassium
iodoacet
to
whole
is
gly-
recommended
of sodium
iodoacetate
be
was
investigated
AND/OR
ON
TIlE
iactic
since
in
added
to whole
blood
of 2.5 mg.
of sodium
is
conimonly
; time lactic
I000ACETATE
WHOLE
decrease
explained,
acid
PREVF.N
used
to
concentration
THIN
OF
GLYCOLYSIS
Bi.ooo
blood
fluoride
conditions
Change
-
Temperature
(CC.)
mg/mt.
0
blood
prevent
(15)
which
Sodium
ate
mg/nit.
10 mg.
oxalate,
Storage
Sodium
col-
the
would
the iodoacetate
was
solutions.
A mixture
IN
Presersotive
whichi
and
after
because
when
(oldfarb
It
to
inconvenient
present
and
blood.
present
immediately
often
system
fluoride
of drawn
preservative
1)100(1 was
responsible
for a 7-31%
fall in lactic
acid levels
could
not
2 mg.
blood
Table
Chemistry
of blood
as a preservative.
The
present
investigation
decrease
in lactic
acid concentrations
with
this mixture
simown in Table
4, the use of 10 mg. of iodoacetate
alone
time drop
occurred
but not to lactic
fluoride
content
some
procedure
of 10 mug, of sodium
milliliter
levels.
acid
have
precipitate
that
Ier
milliliter
showed!
a slow
(Table
4). As
lactic
to
latter
it requires
dl rawii.
A. search
a mixture
tue
either
or
blood.
for
Clinical
Samples
therefore,
inlmibit
& KAPLAN
0
Time
Lactic
im,id
concentration
(hr.)
1.11
acid
concentration
(%)
(aiM)
0
in
tactic
(Reference
sample)
10
10
10
0
0
2.5
4
4
S
24
0.90
0.90
25
8
1.03
4
8
0.93
-
16
7
-
24
0.77
-
31
4
24
0.98
-
12
25
8
1.30
+
Ill
4
8
1.33
+20
8
24
1.21
2.44
+
25
24
1.23
24
1.21
+
+
8
24
1.11
1.12
+
-20
4
10
-
25
-10
0
19
19
-
4
4
9
+120
11
9
0
1
Vol.
12, No.
9,
increased!
1966
LACTIC
20%
by
eoncentratioll
refrigerated
4, 2 bottom
Therefore,
was
oxalate
for
at
of
per
completely
refrigerator
IN
8 1w. at
to
10
no
rise
569
BLOOD
4#{176}.\Vlien
nig./ml.
ill
the
acid
lactic
sodium
fluoride
and
time sample
blood
of
in 8 hr.
or
in
24 hr.
lines).
a mixture
potassium
for
iiicreased
at 4#{176},
timere
(Table
colysis
staliding
on
was
ACID
10
the
of
ing.
milliliter
of
indicated
sodium
blood
time
fluoride
is
and
adequate
when
the
to
sample
2
iiig.
of
inhibit
gly-
is stored
in
a
40,
Conclusion
The
nietiiod
described
terminatioii
chromatograph.
this
iii
report
of blood
lactic
acid
Many
different
be used
since
phase
injection
sensitivity
after
eerie
tile
tamination
and
The collection
niakes
levels
types
possible
for laboratories
of instruments
required
sulfate
time
possessnig
and detectors
is relatively
low.
oxidlatioll
The
glycolysis
analyses
may
as well
be
as clotting
performed
at
the
when
stored
convenience
of
vapor
con-
column
oxalate
at
dea gas
may
use
eliminates
some
potential
interfering
compounds.
of blood
in a sodium
fluoride-potassium
prevents
raj)idl
mixture
subsequent
and
40,
of time analyst.
References
1.
Huckabee,
2.
3.
Tranquada,
R. E., Lactic
acidosis.
A review.
Calif.
lied.
101,
450
Pfleiclerer,
G., and
I)ose,
K.,
Eine
euzyniatisclie
Bestimniung
der
Milchsiiuredeliydrase.
Biochem.
Z. 326,
436
(1955).
Loomis,
M. E., An enzymatic
fluorometrie
method
for
the determination
serum.
J Lab.
Cliii.
Mcd.
57, 966 (1961).
Olson,
G. F., Optimal
eoIi(litjoIls
for
the
cnzylliatic
lleterlninhitiOn
Chein.
8, 1 (1962).
Parijs,
J., and Bnrbiei,
F., The enzymatic
L (+)
lactate
ieterniination
Chem.
3, 74 (1965).
Hochella
,
N. J., and
Weilillonse,
S .,A utoma
ted
lactic
or iii deterni
tissue
extracts.
Anal.
Biochem.
10, 31)4 (1965).
4.
5.
6.
7.
8.
W
Barker,
S.
logical
9.
10.
11.
13.
14.
15.
S.
B.,
D.,
Friedemann,
J.ong,
40,
12.
B.,
material.
Barker,
Seligson,
100,
.
E.,
Alinoiiual
method
Natelsoll,
ponents
acetolie,
Maass,
0.,
temperature
pounds
Bueding,
glycolysis
N.
blood
.Ini.
lactate
and
Suiiimersoii,
W. II.,
dolorimetric
J. Riot.
Chem.
138,
535
(1941).
“Lactic
Acid.”
In
Standard
Methods
Ed.
Academic
Press,
New
York,
1961,
T. E., and
291
(1933).
C., The
stabilization
27 (1946).
Hoffman,
resting
Graeser,
and
.1.
B.,
estimation
The
.J.
determination
p.
of
Clinical
167.
determination
of
lactic
30,
Mcd.
of
acid
III
lactic.
833,
(1961).
(1964).
L(+)-Milehs#{228}ure
of
of
lactic
L-lactic
in
i nat
ion
of
lactic
1)10011 samples.
acid
blood.
in
Z.
hIm.
serum
acid
and
in
(Vol.
.1.
in
Cliii.
acid.
Chemistry
arid.
mit
Bioi
Bioehem.
bio3),
Chem.
.J.
E., Barhoriak,
J. J., nail Ilardlnan,
11. F., A sensitive
gas
chromatographic
for the determination
of lactic
acid.
A no!. Biochem.
9, 175
(1964).
S., and
Stellate,
R. L., Tastrimientation
for
the
concentration
of trace
comof a mixture
for
gas
chromatography
.
Application
to the
determination
of
ethanol,
methanol,
and
2-propanol
in hi ood.
Microeheni.
J. 9, 215
(1965).
and
Boomei,
E. H.,
Vapor
(Iensitws
at two
pressures
1111(1 over
an extended
ralige.
I. The
properties
of ctlieylene
oxide
compared
to oxygen
comof similar
molecular
weight.
.J. A iii. Client.
Soc.
44,
1709
(1922).
E., and
Goldfarb,
W.,
The
effect
of sodium
fluoride
and
sodiuln
iodoacetate
on
in human
blood.
J. Blot.
Chem.
141,
539
(1941).