Modified Enzymatic Methods for the Determination
of L-(+)-Lactic and Pyruvic Acids in Blood
J. F. NEVILLE, JR., M.D., AND R. L. GELDER, CH(ASCP)
Department of Surgery, Cardiovascular Laboratory, Upstate Medical
State University of New York, Syracuse, New York 13210
Center,
ABSTRACT
Neville, J. F., Jr., and Gelder, R. L.: Modified enzymatic methods for the determination of L-(+)-lactic and pyruvic acids in blood. Amer. J. Clin. Path. 55:
152-158, 1971. Modifications of the enzymatic methods of analyses for L-(+)lactic and pyruvic acids in whole blood result in average recoveries of 99% for
lactate and 100% for pyruvate. The modified lactate procedure facilitates
the handling of a large number of analyses. The pyruvate blood recovery
studies demonstrated that proper centrifugation following deproteinization is
a prime determinant for achieving 100% recovery from whole blood. The
importance of technic in performing recovery experiments is emphasized.
ENZYMATIC METHODS for analyses of L-(+)lactic 3> *•a'8'13"15 and pyruvic *> 2<5"7' 12 >"- 1B
acids in biologic materials have been described by various authors. In our investigations of anaerobic metabolism in dogs, we
encountered the following problems in analyzing whole blood for these metabolites:
nonlinear results were obtained with pyruvate standards; the pyruvate analyses failed
to yield quantitative recoveries; the analytical range for lactate was inadequate for
the levels to be measured; when large numbers of lactate analyses were to be performed, the time required for multiple
pipettings and spectrophotometric readings
became prohibitive.
We have modified the basic technics to
obviate these problems.
relationship:
LDH
Lactate + NAD+ *
I n the lactate analysis, conducted at an
alkaline pH, the original pyruvate present
is removed as the hydrazone or semicarbazide. Subsequent addition of LDH drives
the reaction to the right, and continued removal of the pyruvate results in all of the
lactate's being consumed, with an equimolar generation of NADH from N A D \
In the pyruvate analysis, by using a neutral pH, the addition of LDH converts all
of the pyruvate to lactate with an equimolar loss of NADH.
By measuring the change in NADH concentration spectrophotometrically at 340
nm., the corresponding lactate and pyruvate concentrations can be calculated.
Materials and Methods
Principle
Enzymatic methods for lactate:pyruvate
determinations depend upon the following
Received January 14, 1970; accepted for publication May 5, 1970.
Supported by USPHS Grant HE-07093-02.
NADH + H+ + pyruteva
Preliminary
Remarks
If blood is allowed to stand for a short
time, changes in pyruvate and lactate will
occur due to glycolytic activity of the erythrocytes.10 Therefore, free flow of blood into
chilled perchloric acid is the preferred
method of sampling.
152
February 1971
DETERMINATION OF LACTIC AND PYRUVIC ACIDS
Our results are expressed as millimoles
per liter of blood water,10 which is calculated from the change in weight ® of a
sample of blood following heating at 105 C.
overnight.
T o compensate for changes in absorbance
due to factors other than the enzyme-induced changes in NADH, a reagent blank
of water and perchloric acid is utilized in
both analyses. Centrifugation was carried
out in an International Model 2K centrifuge with an I.E. 811 head at 4,200 X g.
Spectrophotometry readings were taken
with a Beckman DU monochromator,
equipped with a Gilford Instrument Co.
Model 220 Optical Density Converter (wavelength, 340 nm.; light path, 1 cm.).
Reagents for Lactate Analysis
Perchloric acid: (Allied Chemicals, 70%)
ca. 6%. Dilute 50 ml. of 70% HC10 4 to 1
liter.
Glycine buffer: (Fisher Scientific, mol.
wt. = 75.07). 0.5 M glycine, ca. 0.4 M hydrazine (as the hydrate, Arthur H. Thomas,
mol. wt. = 50.064), and 0.0054 M EDTA
(as the disodium salt, dihydrate; Sigma,
99.5%, mol. wt. = 372.24), final pH 9.5.
Dissolve 37.54 Gm. of glycine and 2.0 Gm.
EDTA in ca. 500 ml. water. Add 20 ml. of
hydrazine hydrate and mix; add 100 ml. of
2 N NaOH, mix, and dilute to 1 liter. The
buffer is stable for 7 to 10 clays if kept in
a refrigerator.
LDH: (Sigma Chemical, Type III). Dilute the enzyme with 2.1 M (ca. 33%) ammonium sulfate to a final concentration of
at least 3,000 units.t
Nicotinamide adenine dinucleotide, beta
form (Sigma Chemical, Grade III from
yeast, mol. wt. = 663.0 anhydrous).
KOH: ca. 5 M. Dissolve 70.14 Gm. of
KOH and make up to 250 ml.
Zinc lactate: (Mann Research Laboratories; L-(+)-lactic acid, mol. wt. = 121.775
• G r a m s o£ blood X % blood water per 100 =
ml. of blood water.
f International Units = mg. per ml. protein X
activity (jumoles per min.).11
153
without 12.97% water of hydration), 20.0
mM per 1. stock lactate solution. Dried zinc
lactate, 0.60887 Gm., is dissolved in, and
diluted with, 6% HC10 4 to 250 ml.
Standard solutions: 1.0 to 6.0 mM per 1.
lactate. Make appropriate dilutions of the
stock solution with 6% HC10 4 .
Reagents for Pyruvate Analysis
Perchloric acid: same as described above.
Triethanolamine (TEA) buffer: (Nutritional Biochemicals Corp., mol. wt. =
185.65, the hydrochloride); 0.4 M TEA,
0.004M EDTA; final pH 7.4. Dissolve 18.6
Gm. of TEA in ca. 200 ml. of water; 12.0
ml. of 2 N NaOH is added to this and
mixed. Add 0.37 Gm. of EDTA-N 2 H 2 2 H 2 0 , mix, and dilute to a final volume
of 250 ml. The buffer is stable if kept in
a refrigerator.
NADH: (Sigma, disodium salt, Grade III,
ca. 98%, mol. wt. = 709.0 in anhydrous
form). Dissolve either 1.4 mg. per ml. or
5.0 mg. per ml. in TEA buffer. Make fresh
daily.
LDH (Sigma Chemical, Type III): dilute the enzyme with 2.1 M ammonium sulfate to yield a minimum of ca. 2,500 units.t
store in a refrigerator; during analysis, keep
in an ice bath.
KOH: same as described above.
Sodium pyruvate: (Sigma, Type II, mol.
wt. = 110.0); 2.0 mM per 1. stock pyruvate
solution. Sodium pyruvate, 22.0 mg., is dissolved in, and diluted with, 6% HC10 4 to
100 ml.
Standard solutions: 0.1 to 0.6 mM per 1.
pyruvate. Make appropriate dilutions of
the stock pyruvate solution with 6% HC10 4 .
All reagents used were of analytical
grade; all solutions were made with "Water
for Parenterals" (Abbott Laboratories) unless otherwise stated.
Procedure
A) Standard Preparation. Standard solutions of both lactate and pyruvate were
diluted with water and 6% HC10 4 (1:1:3)
154
NEVILLE AND GELDER
to give a 1/5 dilution of standard to total
volume. A reagent blank of water and 6%
HC10 4 (1:4) was also prepared. The 5.0-ml.
aliquots of standards and blank were subsequently chilled in an ice bath.
B) Blood Deproteinization. Blood samples were obtained by allowing about 2 ml.
of arterial blood to flow into preweighed
centrifuge tubes containing 5.0 ml. of
chilled 6% HCIO4, similar to the methods
of Hohorst 8 and Bucher and associates.2
After the denatured blood and perchlorate
were equilibrated at room temperature, the
tubes were reweighed and 6% HC10 4 was
added X to give an approximate ratio of
blood water to total volume of 1/5. The
tubes were centrifuged at 4,200 x g. for 15
min., and the supernatants were decanted
and recentrifuged for an additional 10 min.
From these supernatants, 5.0 ml. aliquots
were taken and chilled in an ice bath.
C) Partial Neutralization. A predetermined amount of 5 M KOH was added §
to the chilled standard, blank, and the
blood supernatants to yield pH values of
1.6 to 2.5. These were mixed on a vortex
and centrifuged. While still chilled, the
supernatants were decanted into tubes, with
care so as not to resuspend and carry over
the KCIO4 precipitate. The supernatants
were allowed to warm to room temperature.
D) Reaction Mixture for Lactate Analysis. Depending upon the expected number
of analyses, a reaction mixture was prepared just prior to use, as follows:
3.0 ml. of glycine buffer X number of analyses
4.0 mg. of NAD+
X number of analyses
10.0 units of LDH
X number of analyses
^Assuming 80% blood water, to achieve a 1:5
dilution of blood water:perchlorate, multiply 0.8 X
4 x weight o£ blood. This is the total amount of
6% HCIO, needed. T h e total ml. of perchlorate
less original 5.0 ml. = ml. of perchlorate to be
added.
§ T h e exact amounts necessary in the above have
to be determined for each batch of 6% HClOj and
KOH. This is usually about 0.56 ml. for standards
and blank, and about 0.46 ml. for blood supernatants.
A.J.C.P.—Vol.
55
E) Lactate Analysis. 0.3-ml. amounts of
the partially neutralized supernatants (step
C) were pipetted into test tubes. T o each
was added 3.0 ml. of the freshly prepared
reaction mixture. The solutions were mixed
and incubated at 37 C. for 1 hr. After remixing, the contents of each unknown and
standard were transferred to a cuvette and
the absorbance (E2) measured. T h e absorbance of the blank (E,) was remeasured
with each set of unknowns. All analyses, including the blank, were done in duplicate.
F) Pyruvate Analysis. The following were
pipetted H into 10 by 40 mm. cuvettes in
the given order:
2.0 ml. of supernatant (step C)
1.0 ml. of TEA buffer
0.1 ml. of NADH solution
The contents of the cuvettes were mixed
with a stirring rod II and the Ej^ absorbance
read after about 2 min. The readings were
repeated until a stable value was obtained.
The reaction was started by the addition
and mixing of 0.01 ml. of LDH in each
cuvette. Starting about 3 to 4 min. after
the addition of LDH, the E 2 absorbance
was read until a stable value was achieved.
The reagent blank was analyzed in order
to determine the absorbance change due to
the dilution by LDH of the Et value. This
is subtracted from the AE of the unknowns
and standards to eliminate a systematic
error in calculations.
G) Calculations.
mM lactate: pyruvate per liter = AE X K
where:
lactates
Ei = absorbance of reagent blank
Ej = absorbance of the unknown
AE = E2 — Ei
_,
dilution factor
K
O^d
pyruvates
Ei = absorbance before addition of L D H
E2 = absorbance after addition of LDH
AE = (Ei — E2)-correction of blank
fl T h e volumetric additions of NADH and LDH
were carried out using Hamilton Co. Microliter
syringes with Chaney adaptors.
|| Polypropylene cell stirring rod with flared end,
Kopp Scientific, Inc.
February
1971
155
DETERMINATION OF LACTIC AND PYRUVIC ACIDS
and:
X*
e = extinction coefficient of NADH at 340 nm.
(6.22 sq. cm. per /aM).9
d = light path of the cuvette, 1 cm.
dilution factor = reciprocal of all dilutions.
Example
of the Pyruvate
1) Original volume of HClOi
2) Weight of blood added
3) Total HC10 4
= 0.8 X 4 X 3.000
4) Total volume
= 9.6 + (0.8 X 3.000)
5) Blood water dilution
= 2.4/12.0
6) Neutralization dilution
7) Analysis dilution
= (2.0 + 0.1 + 0.01)
8) K = 5/1 X 5.46/5
X 3.11/2 X 1/6.22
9) Upon analysis:
Calculations
=
=
5.0 ml.
3.000 Cm.
=
9.6 ml.
9.0 m M / 1 . s t a n d a r d
.800
.700
= 12.0
=
=
1/5
5.0/5.46 (blood)
5.0/5.56 (standard)
=
2.0/3.11
=
=
5.45:4
1.365 for a blood
factor.
Ei == 1.300 absorbance units
E j == 0.975 absorbance units
.600
Z
. 50C
m
O
'SI
(0
<•
.400
AE == 0.325 absorbance units
and correction of blank = 0.003 absorbance units
1.0 m M / 1 . s t a n d a r d
corrected AE = 0.322 absorbance units
10) Corrected AE X factor = mM pyruvate per
1. blood water
0.322 X 1.365 = 0.440 mM pyruvate per
1. blood water.
Quality Control: T h e percentage recovery of the standards, which were analyzed
in conjunction with the blood unknowns,
was the quality control of the procedures.
m
% recovery =
Conversion
mM of lactate/pyruvate obtained . . .„„
'-^%r ,,
; X 100
mM of lactate/pyruvate expected
of mM to mg. per 100 ml.
mg. per 100 ml. pyruvate
8.806 X mM per 1. pyruvate.
mg. per 100 ml. lactate
•• 9.008 X mM per 1. lactate.
Results
Lactates
Zinc lactate standards (0.5 to 6.0 m M
per 1. lactate before dilution) in 6 % H C 1 0 4
yielded an average recovery of 100.1%, (N
= 93, SD = 3 . 1 % ) . I n blood recovery studies, blood was added to perchlorate containing known amounts of lactate; an average recovery of 99.0% (N = 24, SD = 2.6%)
was obtained.
i_L
TIME
FIG. 1. Rate of absorbance change in the reagent
blank and lactate standards (9.0 mM and 1.0 mM)
vs. time. Partially neutralized supernatant (0.3 ml.;
pH 1.6 to 2.5 with 5 M KOH) plus 3.0 ml. of reaction mixture.
W h e n lactate is the only metabolite to be
analyzed, the deproteinization ratio of
blood water to the total volume may be
adjusted to a smaller r a t i o t h a n used here,
so long as the a m o u n t of K O H added is
adjusted to m a i n t a i n the p H of the supern a t a n t i n the range of 1.6 to 2.5 prior to
addition of the reaction mixture. W e have
used 0.1 to 0.5-ml. aliquots of s u p e r n a t a n t
i n the m e t h o d described, as well as de-
156
A.J.C.P.—Vol.
NEVILLE AND GELDER
Table 1. Percentage Recoveries of Pyruvate
Standards in 6% HC104*
mM per 1. 5 M K 2 C0 3
102.0
96.8
93.9
93.1
0.10
0.20
0.40
0.60
5 M K 3 PO,
5 M KOH
106.0
100.0
102.5
84.5
99.9
99.6
101.4
100.1
* Standard supernatants partially neutralized to pH
values of 1.6 to 2.5 with various 5 M K + reagents.
proteinization ratios of both 1/5 and 1/4,
without bias in recovery or loss of linearity.
We found from time studies that by
using sufficient excess LDH (> 15 units per
reaction mixture tube), the analysis could
be completed within an incubation period
of an hour for lactate concentrations as
high as 9.0 mM per 1. (Fig. 1). Using hydrazine sulfate instead of the hydrate or decreasing the molarity of the hydrazine by
half did not appreciably decrease the absorbance of the reagent blank. However,
when the NAD + concentration was halved,
the recovery of the higher concentrations
of lactate was suppressed.
Pyruvates
Utilizing the method described, sodium
pyruvate standards (0.1 to 0.6 mM pyruvate
before dilution) in 6% HCIO4 yielded an
average recovery of 100.2% (N = 123, SD =
1.4%). In blood recovery studies, blood was
added to perchlorate containing known
amounts of pyruvate; the average recovery
was 100.1% (N = 26, SD = 2.6%).
55
Standards. When either 5 M K 3 P0 4 or
5 M K 2 CO s was used for neutralization, the
percentage recoveries of the standards decreased as the concentration of the pyruvate increased (Table 1).
When we used 0.04 M EDTA in the 0.4
M TEA buffer,2 the percentage recoveries
of the pyruvate standards decreased as
the concentrations of standards increased
(Table 2). Using 0.4 M TEA without
EDTA, 0.4 M TEA with 0.004 M EDTA,
and 0.4 M Tris" linear and quantitative recoveries of standard were obtained, provided
that the pH of the supernatant after addition of KOH was in the range of 1.6 to 2.5.
B'.ood Recoveries. When recovery experiments used pyruvate added to plasma or to
deproteinized supernatants, 100% recovery
was achieved routinely. Addition of the
pyruvate to the HCIO4 used for deproteinization resulted in only 80 to 90% recovery,
which could not be improved by different
methods of deproteinization. 3 - 5 - ° It was
noted that the radial centrifugal force of
our centrifugation was lower than recommended. 2 Subsequent centrifugation of the
blood perchlorate mixture at 4,200 X g. or
more corrected this problem.
Re-examination of K 3 P 0 4 vs. KOH for
use in the partial neutralization step of
analyzing blood supernatants was undertaken. Recovery of pyruvate utilizing
K 3 P0 4 was in the 80 to 90% range, whereas
" T r i s = 2-amino-2-(hydroxymethyl)-l,3-propancdiol, Eastman Organic.
Table 2. Percentage Recoveries of Pyruvate Standards in 6% HC10V
mM per 1.
0.05
0.10
0.15
0.20
0.30
0.40
0.60
0.4 M TEA
0.04 M EDTA
104.0
96.0
94.3
92.8
0.4 M TEA
0.004 M EDTA
0.4 M TEA
0.4 M Tris
99.9
100.7
101.5
98.8
99.9
100.1
101.2
99.2
100.6
97.8
100.1
98.6
99.4
100.8
* Standard supernatants partially neutralized with 5 M KOH to p H values of 1.6 to 2.5 and analyzed utilizing
various buffers.
February 1971
DETERMINATION OF LACTIC AND PYRUVIC ACIDS
157
0.60
FIG. 2. Recovery of pyruvate from blood after
partial neutralization of
blood supernatants with
either 5 M K3PO< or 5 M
KOH. KOH neutralization yielded an average
recovery of 100%; K 3 P0 4
neutralization yielded 80
to 90% recovery.
0.20
0.10
0.20
0.30
0.40
0.60
rail Pyruvate Expected
100% recovery was achieved utilizing KOH
(Fig. 2).
Discussion
Lactates
The reaction mixture technic for lactate
analysis was developed primarily because
of the large numbers of samples which we
were analyzing and the extended range of
analysis that we needed.
Validity of the reaction mixture is predicated upon the assumption that the reagent blank (containing NAD-LDH-hydrazine, with an absorbance in itself) changes
at the same rate as in the unknown sample,
independent of the absorbance change due
to the conversion of NAD + to NADH (Fig.
1). Lundholm and colleagues 13 observed
that as the lactate concentration in the cuvette increased, the pH of the reaction solution fell, due to an increase of H + ions
as the lactate was oxidized to pyruvate.
This subsequently led to a difference be-
tween the absorption of the blank and that
of the unknown, giving high recoveries. We
have not observed this phenomenon, perhaps because we partially neutralize the
supernatant prior to analysis and include
EDTA in the buffer.
For lactates, the enzyme and coenzyme
are in excess. The procedure as described
permits large A absorbance per mM of lactate (resulting from a small dilution factor),
thereby minimizing analytical errors.
We have only suggestive evidence that
recovery of lactate from blood is dependent
upon high centrifugation speeds. Hohorst 8
mentions a minimum of 3,000 X g.
Pyruvate
The original discrepancy between recoveries when pyruvate was in the perchlorate
used to precipitate protein and when pyruvate was added to the protein-free supernatant emphasizes the need for proper controls in evaluating an analytical method. It
158
NEVILLE AND GELDER
is not valid to assume that the supernatant
contains all of the original substrate just
because substrate added to the supernatant
can be recovered quantitatively.
Closter and Harris, 5 and Landon and associates 12 have offered the view that low
recoveries, or inability to recover pyruvate
added to the perchloric acid prior to denaturization, were due to the inability to
deproteinize the blood completely and
thereby inactivate the LDH. We consistently had quantitative recoveries of pyruvate added to blood supernatants which
would not have been possible with residual
LDH present.
Our studies indicate that the need for
centrifugation at high radial centrifugal
force cannot be overemphasized as part of
the pyruvate analysis. This may be the
reason that Gloster and Harris 5 failed to
obtain accurate blood recoveries when perchloric acid was used as the deproteinizing
agent.
Using 5 mg. per ml. of NADH in the
pyruvate analysis, the Ex value lies between
1.100 and 1.300 absorbance units, permitting a wide range of pyruvate to be analyzed. The small dilution factors provide a
large A absorbance per mM pyruvate and
increase the accuracy of a given determination.
For those spectrophotometers in which
accuracy of reading above a 0.500 absorbance is a factor, the concentrations of
NADH for pyruvate analysis and NAD+ for
lactate analysis must be decreased. The dilution of supernatants will have to be increased to encompass the same range of
unknowns.
A.J.C.P.—Vol.
55
Acknowledgment.
Mr. Donell Bacon and Mrs.
Anna Marie Fehling gave generous interest and
technical assistance.
References
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In pamphlet "Enzymatic determination of the
pyruvate concentration in blood. ETC-C973,
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Boehringer & Soehne, Mannheim, West Germany, 1961.
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