The Determination of Lactic Acid in Microgram

Quantities
BY R. P. HULLIN AND R. L. NOBLE
Department of Biochemi8try, Univer8ity of Leeds
(Received 13 February 1953)
The conversion of lactic acid to acetaldehyde by hot concentrated
sulphuric acid was first reported by Deniges (1909) and its use as. a
basis for the determination of lactic acid was described by Mendel &
Goldscheider (1925). Eegriwe (1933) described a reaction between p-
hydroxydiphenyl and acetaldehyde to yield a violet colour and this
colour reaction was first adopted as a sensitive means of estimating
lactic acid by Miller & Muntz (1938).
The method most widely employed for determining lactic acid on a
microscale is that ofBarker & Sunmmerson (1941). In the course of
work in this laboratory requiring the estimation of very small
amounts of lactic acid, usually in the presence of pyruvate,
reproducible results could not be achieved with this technique;
further, the final -optical densities were not directly
proportional to -the amounts of lactic acid present.This paper
contains the results of our investigations whereby several
modifications of Barker & Siummerson's procedure were made. With
these changes, 1-8 ,ug. of lactic acid could be determined
with an accuracy of ± 2%. When interfering pyruvic acid is present,
the procedure involves a dilution so that the method in this case is
applicable to 2-10 pig. lactic acid.
METHODS
ReagentM
p-Hydroxydiphenyl (1.5 g.) is dissolved in 10 ml. of 5% (w/v) NaOH and
diluted to 100 ml. with water.
Standard lactic acid solution: 0-2133 g. of pure, dry lithium lactate
(Hillig, 1937), was dissolved in about 100 ml.of water, 1 ml. of
concentrated H2SO4 (A.R.) added and the solution made up to 1 1. with
water.
Recommended method
The protein-free solution (2 ml.) containing 10-80 pg. of lactic acid, is
pipetted into a 150 x 25 mm. test tube which contains 1 ml. of 20% (w/v)
CUS04, 5H20 and the final volume is made up to 10 ml. with water.
Approximately 1 g. of solid Ca(OH)2 is added and, after thoroughly mixing
and allowing to stand for 30 min. or more, the solution is centrifuged.
If the original 2 ml. of protein-free solution contains 100-60 ptg. of
pyruvic acid, 6 ml. of the supernatant solution from the first treatment
with copper-lime is pipetted into a second tube containing 0-6 ml. of 20%
(w/v) CUS04, 5H20 and 0-6 g. of solid Ca(OH), added. This procedure is
then repeated a third time with 3 ml. of supernatant solution,0-3 ml. of
20% CUS04, 5H20 and 0-3 g. of Ca(OH)2,whereby the amount of pyruvic acid
present is reduced to a minimum and the colour interference due to it
becomes a small constant value which may be deducted from the
final optical density.1 ml. of the supernatant solution from the third
copperlime treatment is transferred to another 150 x 25 mm. test
tube, held in the arm of a mechanical shaker with its lower end immersed
in an ice-water mixture. CuS04, 5H20(0.05 ml. of 12% (w/v) solution) is
added followed by 6 ml.of conc. H2SO4 (A.R.) dropwise from a burette with
vigorous shaking, the tap of the burette being lubricated with the
concentrated acid. After complete addition, the contents are poured and
allowed to drain into a small ground-glass stoppered Pyrex tube which is
then heated for 30 min. in a water bath maintained at 60+ 10. The tube is
allowed to cool to 10-15°, the stopper removed, 0-1 ml. of the
p-hydroxydiphenyl reagent added and the precipitated p-hydroxydiphenyl
thoroughly dispersed in the H2S04. Incubation of the tube for 20 min. at
28-30° to ensure maximum colour development is then followed by 90 sec. in
a boiling-water bath to destroy excess p-hydroxydiphenyl.After this
treatment the tube is immediately cooled in icewater and the optical
density ofthe resulting violet-coloured solution is determined in circular
tubes of 1 cm. diameter with the Unicam diffraction-grating
spectrophotometer at a wavelength peak of 560 m,u. against a reagent
blank prepared by taking distilled water through the whole of the
above procedure.The calibration curve obtained by this method (E =,Ag.
lactic acid x 0 059) for 0-8 ,ug. of lactic acid is a straight line.
DEVELOPMENT OF METHOD
The effect of varying the experimental conditions at different stages of
the method is described in Table 1. The absorption curve of the
acetaldehyde-p-hydroxydiphenyl complex shows a sharp maximum at 560
m,t. as illustrated by the following optical densities obtained from
6,ug. Of lactic acid in the conditions described on p. 289 at wavelengths
between 520 and 600 mp.: 520, 0-210; 540, 0-295; 550, 0.330; 560, 0
345; 570, 0-330; 580, 0-305; 600, 0-185.
The calibration curve, which is strictly reproducible, indicates
that the coloured complex obeys the Lambert-Beer Law in the concentration
range of 1-8,ug. of lactic acid.Above 8 pg. the optical density does
not increase in direct proportionality with the amount of lactic acid but
falls off slightly. If coloured solutions, obtained from larger quantities
of lactic acid (up to 10,ug.), are diluted with H2SO4:water (6:1, v/v)
the optical densities of the diluted solutions are directly proportional to
the amounts of lactic acid originally present.Speciflcity of the method
The work for which the method has been used required the determination of
lactic acid in the presence of acetoin, butane-2:3-diol, diacetyl and
pyruvate. None of the first three of these compounds gives any colour when
the method is appliedto solutions containing 60.ug. ofit and quantitative
recoveries of lactic acid are always obtained in the presence of such
amounts. However, pyruvic acid, which is commonly present with lactic acid
in media from enzymic studies,seriously interferes with the reproducibility
of the method unless steps are taken to remove it. This is illustrated by
the results collected in Table 2 for pyruvic acid samples without
copper-lime treatment. One copper-lime treatment does not yield
reproducible results, although it clearly removes a considerable amount of
pyruvic acid. The repetition of the copper-lime procedure seemed a likely
way of completely eliminating the interference due to pyruvate and the
effect of three successive treatments is also illustrated by the results in
Table 2. The third treatment reduces the optical density of the interfering
pyruvic acid to a small constant value. Using this technique, recoveries
oflactic acid ranging from 98 to 102% may be obtained in mixtures
containing 2-10lg. of lactic acid and 10-60kg. of pyruvic acid/ml. of
final solution; Table 1. Effects of varying experimental conditions
('Colour' means optical density at 560 myt.)
Stage Oxidation
Oxidation
Oxidation
Oxidation
Incubation with p-hydroxydiphenyl
reagent
Destruction of excess
p-hydroxydiphenyl
Condition varied
Heating at 1000 in open
tube
Stoppered and unstoppered
tubes
Time of heating at 600
Concn. of Cu2+ added
Time at 28-30O
Time of heating at 100°
Recommended procedure
Stoppered tubes
30 min.
0-05 mhl. of 12% (w/v)
CUS04, 5H20
20 min.
90 sec.
Effect of other conditions The longer the heating, the less colour
obtained Unstoppered tubes give about 15% less colour 24-65 min. gives
maximum colour; <24 min., less colour 12-32%, same colour; <12%,
less colour 20-50 min., same colour; <20 or > 50 min., less colour
30-120 sec., identical colour Table 2. The effect of successive
copper-lime treatments on the colour obtained with pyruvic acid
samples ((I) 9 ml. of standard solutions (containing 200, 300, 400,
500 and 600,g. of pyruvic acid) + 1 ml. of 20% CUS04, 5H2O
+ 1 g. Ca(OH)2. (II) 6 ml. of supernatant solution from (I) +0-6 ml.
20% CU$04, 5H20 +0-6 g. Ca(OH)2. (III) 3 ml. of
supernatant solution from (II) +0-3 ml. 20% CuSO4, 5H20 +0-3 g.
Ca(OH)2. 1 ml. of supematant solution taken for analysis in each case.)
Optical densities (I)
After one copperlime
treatment
0-070
0-105
0-110
0-125
0-130
(II)
After two copperlime
treatments
0-020
0030
0-050
0-050
0-055
(III)
After three copperlime
treatments
0-017
0-020
0-023
0-022
0-020
Pyruvic
acid conen.
(jug./ml. in
final solution)
20
30
40
50
60
Without
treatment
0-200
0-275
0-38
0-43
0-52
290

Table 3. Recoveriea of lactic acidfrom mixture8 of pyruvic and

lactic acid8
Final solution
-A Optical density Recovery of
Lactic acid Pyruvic acid of mixture Optical density lactic acid
(/Ag./ml.) (I.tg./ml.) (Obs.) (Corr.*) (%)
2 60 0*140 0*120 101
4 60 0*265 0.245 102
6 60 0*368 0*348 98
8 60 0'490 0.470 99
6 20 0*385 0*365 103
6 30 0*375 0*355 100
6 40 0*380 0*360 101
6 50 0*368 0-348 98
6 60 0 375 0 355 100
* Observed value minu8 0.02 which is the average optical density
produced by remaiuing pyruvic acid (20-60FIg./ml.
final solution).
these recoveries are corrected for the small constant optical
density due to interfering pyruvic acid after three copperlime
treatments. The results are shown in Table 3.
Similar recoveries are obtained when known quantities of
lactic acid are added to tissue and enzyme extracts and
deproteinization carried out with 10% (w/v) trichloroacetic
acid.
DISCUSSION
The conditions under which the oxidation of lactic
acid to acetaldehyde is carried out, in the first stage
of the method of estimation, are of critical importance.
When this oxidation is carried out in open
tubes, the acetaldehyde recoverable as the final
coloured complex varies greatly with the time of
heating employed. These findings are contrary to
those of Barker & Sunmmerson (1941) who state that
identical results are obtained with heating periods
of 3-10 min. Mendel & Goldscheider (1925), whose
method of lactic acid estimation used 1:2-dimethoxybenzene
instead of p-hydroxydiphenyl to give
a red compound, also claimed that heating in open
tubes for 4-8 min. during the sulphuric acid oxidation
stage produced no differences in the optical
density of the resulting coloured complex. However,
Miller & Muntz (1938) did recommend the use
of stoppered tubes, but gave no reason for this.
Our results confirm those of Barker & Summerson
regarding the influence of copper on the lactic acid
oxidation but we have found it necessary to use
a larger amount (0-05 ml. of 12% CUS04, 5H20
instead of 0'05 ml. of a 4% solution) to achieve the
full increase in sensitivity. This modification may be
required owing to the greater recoveries of lactic
acid as acetaldehyde using stoppered tubes.
The modification ofmethod necessary to estimate
lactic acid accurately in the presence ofpyruvic acid
was suggested by the work of Van Slyke (1917).
Miller & Muntz (1938) have suggested heating the
sulphuric acid oxidation mixture for 15 min.
instead of 5 min. in order to remove any interfering
pyruvate.
SUMMARY
1. Lactic acid has been determined in the range
0-8 ,ug./ml. with an accuracy of ± 2%.
2. The method, which can be employed over a
2-10 jtg./ml. range in the presence of 10-60 ,ug./ml.
of pyruvic acid, is based on that of Barker &
Summerson (1941), but certain modifications are
incorporated to achieve reproducibility.
We wish to thank the Medical Research Coun,cil for
financial amistance, and for a grant to one of us (R. L. N.).
REFERENCES
Barker, S. B. & Summerson, W. H. (1941). J. biol. Chem. 138,
535.
Denig6s, G. (1909). Bull. Soc. chim. Fr. 5, 647.
Eegriwe, E. (1933). Z. anal. Chem. 95, 323.
Hiflig, F. (1937). J. A8s. off. agric. Chem., Wash., 20, 130.
Mendel, B. & Goldscheider, I. (1925). Biochem. Z. 164,163.
Miller, B. F. & Muntz, J. A. (1938). J. biol. Chem. 126,413.
Van Slyke, D. D. (1917). J. biol. Chem. 32, 455.
19-2
292 NOTES AND COMMENT
cells. I. The bleaching process. Biochim. -, AND R. F. SCAGEL. 1958. Diurnal study
Biophys. Act., 29: 359-368. of phytoplankton pigments. An in situ study
STRICKLAND, J. D. H., AND T. R. PARSONS. 1960. in East Sound, Washington. J. Mar. Res.,
A manual of sea water analysis. Bull. Fish. 17 : 567-583.
Res. Bd. Canada, No. 1.25. 185 pp.
YENTSCH. C. S.. AND T. H. RYTHER. 1957. Short C. D. MCALLISTER
term variations in phytoplankton chlorophyll
and their significance. Limnol. Oceanogr., Fisheries Research Board of Canada,
2: 140-142. Nanaimo, B. C.
ESTIMATION OF LACTIC ACID IN SEA WATER SOLUTIONS AND HOMOGENATES
Lactic acid is commonly estimated by the method of Barker and Summerson ( 1941) ; after
oxidation in strong sulphuric acid solution to acetaldehyde the latter is coupled with p-
hydroxydiphenyl in the presence of cupric ions to yield a purple compound. In biological material
protein must be first precipitated, usually with trichloracetic acid, after which other interfering
substances are removed by treatment with copper sulphate and calcium hydroxide. It has
recently been pointed out ( Barnes, Finlayson, and Piatigorsky 1963) that the method is not
entirely satisfactory in strong salt solutions and even less so in sea water of normal salinity
(+ 32%0). In the investigation of anaerobic processes in marine animals the estimation
of lactic acid in sea water media is often of considerable importance and a modification
of the Barker and Summerson method has, therefore, been developed which is
applicable in such media.Reagents, apparatus, and methods (i ) Copper sulphate: B.D.H.,
Analytical Reagent grade,
(ii) Calcium hydroxide: B.D.H., finely precipitated,
(iii) Sulphuric acid: B.D.H., Analytical Reagent grade, nitrogen-free,
(iv) p-hydroxydiphenyl: a commercial product recrystallized several times,
(v) Thallous sulphate: B.D.H., Analytical Reagent grade,
(vi) Trichloracetic acid: B.D.H., Analytical Reagent grade,
(vii) Lactic acid standards: pure recrystallized lithium lactate.
The reaction was carried out with the slight modifications recommended in Umbreit,
Burris, and Stauffer ( 1945; p 104), the transmittances being measured at 560
rnp on a Unicam spectrophotometer. Concentrations are given in pg/ml of the solution,
1 ml of which is eventually taken for oxidation and development of the color.
When testing the method on sea water homogenates of biological material, protein
was precipitated with 10% trichloracetic acid and other interfering materials
by the standard copper sulphate-calcium hydroxide treatment.Effect of salt solutions and sea
water Figure 1 gives a number of calibration curves in distilled water, in sodium chloride
solutions, and in sea water, using the unmodified technique, It is evident that, contrary to what
had been anticipated, sodium chloride at a strength equivalent to sea water does not give similar
values; it had previously been thought that the formation of hydrochloric acid when sulphuric
acid is added at the oxidation stage was responsible for the discrepancy. The values in sodium
chloride solutions are clearly less reliable and for this the formation of hydrochloric acid may be
responsible. The reaction is slightly less sensitive in chloride solutions; in sea water it is
much less sensitive and the calibration curves and estimations of unknowns are not satisfactory.
Absorption curves in the various media showed that in all the colored product is identical, and the
lower sensitivity in sea water would therefore seem to be due to inhibition of its develNOTES
AND COMMENT 293 Lithium lactate /uq per ml. FIG. 1. Calibration curves for lactic acid estimation
in various media: (a) Distilled water, (b) 3.5% sodium chloride solution, (c) 0.01% potassium bromide
solution, (d) 50% sea water, (e) sea water. opment or its destruction. In view of the wide variety of
substances tested for interference by Barker and Summerson and a knowledge of the
composition of sea water it seemed that only the bromide content of the latter was likely to be
responsible for the observed results, possibly as a result of the liberation of hydrobromic
acid. Figure 1 shows the effect of adding bromide alone to distilled water, in an equivalent
quantity to that in sea water. The similarity to the calibration curve in sea water alone is striking
and together with the fact that removal of bromide (see below) gives values equivalent to those in
distilled water strongly supports this view. Since the object was only to develop a satisfactory
method for the estimation of lactic acid in sea water, the reasons for the interference have not
been further investigated. Removal of bromide (and some chloride) by thallous sulphate
Krogh and Keys (1934) found that thallous sulphate was a useful reagent with which to remove
chloride from sea water prior to the estimation of its organic carbon content. The solubility of
thallous bromide is only about one-sixth that of the chloride and addition of thallous sulphate
might be expected to serve the present purpose, provided that the presence of thallium ions does
not interfere with any other part of the estimation. Table 1 shows the effect of adding various
quantities of a saturated solution of thallous sulphate to sea water containing lactate.
There is a suggestion in Table 1 that 0.5 ml of saturated thallous sulphate gives somewhat low
returns at the highest lactic acid content; otherwise the method seems adequate to prevent
interference by substances in the sea water and to suppress the variability associated with
solutions containing chloride. Estimation of lactic acid in sea water In view of the above results
the following procedure was adopted for the estimation in solutions of sea water when protein
and other materials are only likely to be present in trace amounts; such is often the case when
any excretion of lactic acid into the surrounding sea water takes place after animals are exposed
to anaerobic conditions.
To 4 ml of the solution add 2 ml of a saturated solution of thallous sulphate. Centrifuge, take 1 ml
of the supernatant, and proceed as described in the Barker-
TABLE 1. Effect of the addition of thallous
sulphate on lactic acid estimation. 2 ml ikctic acid
solution: variable quantity precipitant: results
pg/ml in original lactate solution
Saturated
thallous
sulphate
solution
(ml)
Lactic acid found
(LG.)
0.0 1.1
3.2 5.4 9.6 9.6
0.5 1.1 3.2 5.7 8.6 8.7
1.0 1.2 3.1 5.5 9.6 9.6
2.0 1.1 3.2 5.5 9.5 8.4
294 NOTES AND COMMENT
Summerson method as indicated in Umbreit,
Burris and Stauffer ( 1945).
Estimation when body tissues
present in sea water are If, as often happens, body tissues containing lactic acid must be
homogenized in sea water then the proteins must be removed with trichloracetic acid and other
substances by the copper sulphate-calcium hydroxide treatment, before the addition
of thallous sulphate. The following method was shown to be satisfactory by adding
lactic acid to a tissue homogenate and estimating the return. To 4 ml of the homogenate add 1 ml
of 10% trichloracetic acid (TCA); stir, centrifuge, and transfer the supernatant to a lo-ml standard
flask; wash the precipitate with dilute TCA and add the washings to the supernatant. Neutralize
the solution and make up to 10 ml. Withdraw 4 ml of the neutral solution and add 2 ml of a
saturated solution of thallous sulphate. Centrifuge. Draw off 3 ml of the supernatant and add 0.5
ml of 20% copper sulphate and 0.5 g calcium hydroxide. Allow to stand half an hour with shaking.
Centrifuge. Withdraw 1 ml and proceed as in the Barker-Summerson method. Carry calibration
through whole procedure.Some typical results are given in Table 2.
TABLE 2. Estimation of lactic acid in tissue
homogenates in sea water. For method, see text.
Values are reported as pg/ml lactic added and
found
Lactic acid
found
ha)
1.0 1.1
2.2 2.2
3.1 3.2
4.3 4.3
4.9 4.8
5.4 5.4
6.5 6.4
7.4 7.5
REFERENCES
BARNES, H., D. M. FINLAYSON, AND PIATIGORSKY.
The effect of desiccation and anaerobic conditions
on the behaviour, survival, and general
metabolism of three common cirrepedes. (Zn
press. )
KROGH, A., AND A. B. KEYS. 1934. Methods
for the determination of dissolved organic
carbon and nitrogen in sea water. Biol. Bull.,
67: 132-144.
UMBREXT, W. W., R. H. BURRIS, AND J. F.
STAUFFER. 1945. Manometric techniques
and related methods for the study of tissue
metabolism. Burgess Publ. Co., Minn. 203
PP.
H. BARNES
AND
The Marine Station, D. M. FINLAYSON
Millport, Scotland.
The Estimation of Lactic Acid using Ceric
Sulphate
BY S. R. ELSDEN AND Q. H. GIBSON
Agricultural Research Council Unit of Microbiology, Department of
Microbiology;
and Department of Phy8iology, Univer8ity of Sheffield
(Received 6 March 1954)
Gordon & Quastel (1939) showed that ceric sulphate
oxidizes lactic acid according to the following
equation:
CH3,. CHOH. COOH + 2Ce4+
CH3. CHO + C02 + 2H+ + 2Ce3+,
and they made this reaction the basis of a method
for the estimation of lactic acid. The reaction was
carried out at 500 and the acetaldehyde so formed
was removed by a stream of nitrogen, trapped in
bisulphite and estimated iodometrically in the usual
way. The advantages claimed for this procedure
were simplicity and the fact that very few compounds
gave rise to volatile aldehydes under the
experimental conditions recommended by the
authors. Glucose in particular did not interfere
and a preliminary treatment of glucose-containing
test solutions with the copper-lime reagent was not
considered necessary.
In its essentials the method devised by Long
(1946) was similar to that of Gordon & Quastel
(1939). He showed, however, that ceric sulphate
oxidizes acetaldehyde to acetic acid; but in spite of
this he was able to obtain good yields of acetaldehyde
from lactic acid by aerating very rapidly, thus
removing the acetaldehyde almost as fast as it was
formed. The aeration rate recommended was
500 ml. air/min. as compared with the three to four
bubbles nitrogen/sec. advised by Gordon &
Quastel.
Winnick (1942) carried out the reaction in
Conway vessels with bisulphite in the central
chamber to trap the acetaldehyde and incubated for
2 hr. at 50°. His method was modified by Conway
(1950), who carried out the oxidation in stoppered
test tubes. After 30 min. incubation at 370, he
transferred 1 ml. samples of the reaction mixture
to diffusion vessels where the acetaldehyde was
trapped in bisulphite placed in the centre chamber.
In view of the widely divergent conditions recommended
by the various authors, it seemed desirable,
before using any of the ceric sulphate methods, to
examine the various steps in the procedure further.
The results of these investigations are given below.
EXPERIMENTAL AND RESULTS
Factor8 influencing the oxidation of
lactic acid by ceric 8ulphate
Effect of ceric sulphate concentration and temperature on the
velocity of the reaction. Since carbon dioxide is one of the
products ofthe reaction, Warburg manometers were used to
study the effect of both temperature and concentration of
ceric sulphate on the velocity of the reaction. The reaction
was carried out in N sulphuric acid and 0*2 ml. lithium
lactate solution in N sulphuric acid was added from the side
ESTIMATION OF LACTIC ACID
bulb when thermal equilibrium had been attained. In Fig. 1
are plotted the half times for the reaction at 22, 40 and 53°.
It will be seen that the concentration of ceric sulphate has
a pronounced effect and that at 530 the half time is less than
5 min. even with the most dilute solution of ceric sulphate
tested.
140 r_
120 _
100
E 80
% 60
I

40
20
n
400
lS * I I--

1-2 1-4 1-6 1-8 2-0 2-2 2-4 2-6

Log. m-moles ceric sulphate/l.
Fig. 1. Effect of temperature and ceric sulphate concentration
on the velocity of oxidation of lactic acid. Experiments
were carried out in Warburg manometers. 11-1 -
moles lithium lactate in 0-2 ml. N-H2SO4 were added from
side bulb at zero time; main compartment contained
2-8 ml. ceric sulphate in sulphuric acid; gas phase, air.
1001
90
IV
4,.
E4,
1-2 1-4 1-6 1-8 2-0 2-2 2-4 2-6 2-8
Log. half-time (min.)
Fig. 2. Effect of temperature and ceric sulphate concentration
on the velocity of oxidation of acetaldehyde. Experiments
were carried out in an apparatus similar to that of
Long (1946). Volume of reactants, 10 ml.; each tube
contained 26-2p.moles acetaldehyde; concentration of
sulphuric acid, N. Reaction was stopped by the addition of
excess of ferrous sulphate and residual acetaldehyde
aerated into bisulphite and estimated iodometrically.
Oxidation of aedehyde by ceric Buiphate. Acetaldehyde
and ceric sulphate were incubated in Long's apparatus at
various temperatures and, at suitable time intervals, the
residual ceric sulphate was reduced to the cerous form by
addition of excess of ferrous sulphate. The acetaldehyde
remaining was then transferred by aeration to bisulphite
solution and estimated iodometrically. The test solution
contained 26-2 ltmoles acetaldehyde, and the total volume
including the ceric sulphate solution was 10 ml. The final
concentration of sulphuric acid was normal in all cases. The
resulte are given in Fig. 2 in which the log of the half-time of
acetaldehyde destruction is plotted against temperature
for three different concentrations of ceric sulphate. It will
be seen that the rate of oxidation of acetaldehyde is
greater the higher the temperature and the greater the
ceric sulphate concentration.
Removal of acetaldehyde by aeration. The effects of temperature
and velocity of aeration on the rate of removal of
acetaldehyde were next examined. These experiments were
conducted in an apparatus of the form and dimensions
recommended by Long (1946). The volume of the reaction
mixture was 10 ml. containing 20jsmoles of acetaldehyde in
N sulphuric acid. Air was blown through the mixture and
the acetaldehyde was trapped in bisulphite. The rate of air
flow was measured with a flow meter. After suitable intervals
of time, aeration was stopped and the acetaldehyde trapped
in the bisulphite determined iodometrically. The experiments
were carried out with flows of 200-1000 ml. air/mi.
2-4.-
2-2-
2-0 -

1-8 -

1-6 -

14'-
1X2-
0-8 --\~\
0-6 %-O~
.0-4
0-2
I I I I I 1,
20 30 40 50 60 70 80 90
Temperature (0)
Fig. 3. Effect of rate of air flow and temperature on the
rate of removal of acetaldehyde. Experiments were
carried out in apparatus similar to that of Long (1946).
Each apparatus contained 20pmoles acetaldehyde in
10 ml. N sulphuric acid. Acetaldehyde was trapped in
bisulphite and estimated iodometrically.
V
I I
VoI. 58 155
S. R. ELSDEN AND Q. H. GIBSON
Table 1. Recovery of lactic acid by aeration method
Each reaction tube contained a total volume of 10 ml. mixture which was
normal with respect to H2SO4 and contained
500juequiv. ceric sulphate; T = 600, aeration rate = 500 ml./min., t =
45 min.
Expt. no. ... ... ... ... ...
Zinc lactate added (as pmoles lactic acid)
No. of blanks
No. of estimations
Mean blank value (,tmoles lactic acid)
Mean recovery corrected for blanks
(,umoles lactic acid)
Range
and at temperatures from 20 to 80°. The results are giv
Fig. 3. Over the range studied the rate of remov
acetaldehyde was found to be approximately proport
to (air flow)0 75 and to the vapour pressure of acetalde
which would exist over pure acetaldehyde at the ten
ature of the experiment.
Working conditions for the aeration method. The resu]
the above experiments show that, for a given air flov
creasing either the ceric sulphate concentration or
temperature will diminish the recovery of acetaldel
These experiments have enabled us to define woi
conditions which permit a good recovery of acetalde
from lactic acid. We recommend an initial concentrati
ceric sulphate of 0-05 N, a temperature of 50-60°, an aer
rate of 500-600 ml. air/min./reaction tube and an aeri
time of at least 45 min. Representative recoveries
these conditions are given in Table 1.
Steam distillation method
Although the aeration method gives satisfactory
coveries, the blanks were substantial, due to the contan
tion of the laboratory air with carbonyl compounds
therefore examined alternative ways of carrying oul
reaction. The results described above show that to ol
the greatest rates of formation and the smallest loss
acetaldehyde, it is desirable to work at as high a temperE
as is compatible with the maximum usable rate of aeri
and the minimum concentration of ceric sulphate. S
distillation seemed to offer the best possibility, and
liminary experiments in which the reaction was carries
in the apparatus of Markham (1942) indicated that at
the reaction was complete in a very short time and tha
acetaldehyde could be distilled over into bisulphite in a
10 ml. of distillate. However, losses were encountered
these we considered to be due to the fact that all the
sulphate had to be added at once, with the result that, a
temperature of the mixture, the amount of ceric sulj
present was sufficient to oxidize significant amoun
acetaldehyde. We therefore designed an apparatus in M
it was possible to add the ceric sulphate dropwise whils
steam was passing. The apparatus, which has pr
entirely satisfactory, is shown in Fig. 4. The follo
reagents are required: (i) 0-05N ceric sulphate in N sulp]
acid prepared from a stock solution of 0-5N ceric sulpha
N sulphuric acid. The stock ceric sulphate is standar
with ferrous ammonium sulphate. We have found
sulphate (pure), Hopkin & Williams, to be satisfac
(ii) 1ON sulphuric acid. (iii) 0-5% (w/v) sodium bisul
solution. (iv) 0-1N, 0-O1N and 0-005N iodine solution.
1
5-65
24
1-13
5-39
2
11-3
24
0-87
11-2
3
22-4
4
1-37
22-2
4
112-9
2
4
0-97
109-6
5-00-5-69 10-88-11-91 21-11-22-40 107-9-110-7
,en in
al of
ional 25' ml.
,hyde 20-
aper- 15
Its of 10
v, inityde
~~~yde. ~~~~~~~~~dDoauvbisletyspu
king
Steam condense
hyde Sta int
on of B40 jon
Fig. 4. Apparatus for the estimation of lactic acid by the
steam-distillation method.
The sample containing lactic acid, which preferably
should not exceed 5 ml., is measured into the reaction flask
and sufficient of the lON sulphuric acid added to make the
concentration of this acid normal. The reaction flask is then
attached to the apparatus. The receiving tube which contains
2 ml. of the 0.5% (w/v) sodium bisulphite is attached
in such a way that the tip of the condenser dips below the
surface of the bisulphite solution. The micro-burner
heating the reaction flask is now turned on and the flame
adjusted so that the solution just boils. The steam isthen
turned on and the steam flow adjusted so that 15-20 ml./
min. of distillate is collected (this rapid distillation rate is
essential and necessitates the use of an efficient double
surface condenser); the 0-05N ceric sulphate is then run
dropwise into the reaction flask at such a rate that each drop
is decolorized before the next drop goes in. When a permanent
yellow colour is obtained, indicating that an excess
of ceric sulphate is present, further ceric sulphate up to a
total of 5 ml. is rapidly added. The only critical feature of the
156 I954
ESTIMATION OF LACTIC ACID
Table 2. Recovery of lactic acid by the steam distillation method
In Expt. 1, 0-002574N-12 was used for final titration; blank titration 0
08 ml. 0-002574N-12, and the results corrected
for this amount. In remaining experiments 0 01287N-I2 used and the blank
was too small to be measured.
Expt. no....
No. of estimations
Lactic acid taken (jpmoles)
Mean lactic acid recovered (,umoles)
S.E.
Range
16
2-17
2-14
±0-02
2-08-2-16
2
6
10-85
10-64
±0-05
10-60-10-73
3
6
21-70
21-13
±0-03
21-07-21-14
4
6
32-55
31-82
+0-10
31-61-31-87
estimation is the dropwise addition of ceric sulphate. When
15 ml. of distillate have been collected the receiver is
lowered and distillation is continued until 20 ml. has been
collected. The steam is then discontinued, the microburner
turned out and the receiver placed in an ice-water
bath to cool. While it is cooling, the reaction flask is disconnected
and thoroughly washed out with distilled water.
The still head is also carefully washed to remove traces of
ceric sulphate solution, which, if left, could bring about the
premature oxidation of part of the lactic acid in the next
sample to be analysed.
The acetaldehyde is then estimated in the usual way, using
solid NaHCO3to destroy the aldehyde bisulphite compound.
Like Friedemann & Graeser (1933) we have found that it is
important to cool the mixture thoroughly before the final
titration; the temperature should be 4-5'. The results of a
series of recovery experiments are given in Table 2.
Under the conditions of this method, glucose gives rise to
carbonyl compounds, and we have found it essential to
treat the test solution with the copper-lime reagent; for this
purpose we use 1 ml. 20% (w/v) CUS04, 5H20/10 ml. test
solution and a spatula full of solid Ca(OH)2. This reagent
will also effectively deproteinize bacterial suspensions and
rumen liquor.
Trichloroacetic acid cannot be used, since under the
conditions of the estimation it is oxidized with formation of
such large volumes ofgas that frothing is uncontrollable. The
various methods for the precipitation of proteins which
involve the use of tungstic acid have however proved
entirely satisfactory. We have also found that perchloric
acid (Neuberg, Strauss & Lipkin, 1944) is very satisfactory
where an acid precipitation is required. If perchloric acid
treatment is followed by treatment with the copper-lime
reagent we have found it convenient to add the copper
solution followed by 2N-KOH until a slight trace of cupric
hydroxide is formed, at which point the lime is added. In
this way the perchloric acid is removed as the insoluble
potassium salt. The removal of perchloric acid is not
essential.
Protein hydrolysates, such as are used in bacteriological
media cause some interference, but no carbonyl compounds
were produced from ethanol, from citric acid or from malic
acid.
The results of an experiment in which the lactic acid,
formed from malic acid by Lactobacillus arabinomsu under
the conditions described by Nossal (1951), was estimated
both manometrically and by the steam distillation method
after treatment of the mixture with copper-lime, are given
in Table 3. Table 4 shows the results obtained when a sample
of blood was analysed in triplicate by the steam distillation
method.
Table 3. Estimation of lactic acid formed from DLmalic
acid by the action of the malic decarboxylase
of Lactobacillus arabinosus
The malic decarboxylase converts L-malic acid quantitatively
into lactic acid and CO2, and the amount of CO2
formed is thus a measure of the amount of lactic acid. In
this experiment a solution of DL-malic acid was treated
with the decarboxylase preparation according to the
procedure of Nossal (1951), and at the end of the experiment
the cup contents were transferred quantitatively to a
measuring cylinder, treated with the copper-lime reagent,
made up to 10 ml. and centrifuged; samples of the supernatant
solution were used for the estimation of lactic acid
by the steam distillation method. Strength of iodine,
0-002754N.
Manometric method
Steam distillation method
(a)
(b)
CO2
(,moles)
9-37
Lactic acid
(,umoles)
9-37
9-35
8-92
Table 4. Estimation of lactic acid in defibrinated
sheep's blood by the steam distillation method
Three 2 ml. samples of defibrinated sheep's blood were
taken and 7 ml. of distilled water added to each followed by
2 ml. 15% (wfv) perchloric acid. Precipitates were centrifuged
off and supernatants filtered through cotton wool
into graduated centrifuge tubes; 0-5 ml. 20% (w/v)
CuSO4, 5H20 was added and 40% (w/v) KOH added
dropwise until a slight permanent blue precipitate obtained,
a spatula full of solid Ca(OH)2 (approx. 1 g.)
added, the contents of the tubes were mixed and the
volumes noted. After 30 min. tubes were centrifuged and
5 ml. samples taken for analysis.
Lactic acid
Sample ,umoles/100 ml. blood
123
193
197
200
DISCUSSION
Our observations on the reaction between ceric
sulphate and lactic acid confirm and amplify those
published by Long (1946). Significant amounts of
the acetaldehyde produced are oxidized to acetic
acid if the former is not rapidly removed from the
reaction mixture. The rate of destruction of
acetaldehyde is related to the concentration of ceric
VoI. 58 157
158 S. R. ELSDEN AND Q. H. GIBSON I954
sulphate in the reaction mixture and to the temperature.
Consequently, to obtain maximum recovery
of acetaldehyde, the concentration of ceric sulphate
should be such as to ensure only a slight excess at
the end of the reaction. The rate of aeration should
be the maximum possible. From our results it
would appear that the concentration of ceric sulphate
recommended by Long (1946) is somewhat too
high.
In view of our findings it is difficult to understand
how a quantitative conversion of lactic acid to
acetaldehyde can be obtained by the procedure of
Winnick (1942). This author recommends a mixture
of 3 ml. sample and 1 ml. saturated ceric sulphate
and the reaction is carried out in Conway vessels and
thus relies upon diffusion to remove the acetaldehyde.
Since the purity of his ceric sulphate is not
stated we can only guess at the final concentration,
but it would seem to be in the region of 0 2N.
Likewise the modification of Winnick's procedure
described by Conway (1950) is open to serious
criticism in the light of the observations of both
Long (1946) and ourselves. In this method the
reaction is carried out in stoppered test tubes, under
conditions somewhat similar to those used both by
Long and ourselves to study the oxidation of
acetaldehyde by ceric sulphate.
It is possible to remove the acetaldehyde formed
from lactic acid, almost quantitatively, either by
aeration or by steam distillation, and we have
examined both procedures. We prefer the steam
distillation method for the following reasons. The
blanks are negligible save when using 0-00257N
iodine; but even here they are small and are
equivalent to about 011,mole lactic acid. The
method is extremely rapid, one distillation taking
little more than a minute to perform. The end point
is sharper because the final volume in which the
titration is carried out is only 30 ml. and this is
particularly important when small amounts of
lactic acid are to be estimated. The advantages of
this ceric sulphate method over the permanganate
methods, described by Friedemann, Cotonio &
Shaffer (1927), Friedemann & Kendall (1929), and
Friedemann & Graeser (1933) are speed and the
small volume in which the titration is carried out.
SUMMARY
1. The effect of ceric sulphate concentration and
temperature on the oxidation of lactic acid by ceric
sulphate have been investigated.
2. The effects of temperature and rate of aeration
on the removal of acetaldehyde from solutions have
been investigated.
3. Conditions for the estimation of lactic acid
with ceric sulphate using the aeration method are
described.
4. A new method for the estimation of lactic
acid using ceric sulphate and the removal of
acetaldehyde by steam distillation is described.
The authors wish to thank Mrs M. Martin for technical
assistance and Mr Hatfield for making and helping with the
design of the apparatus.
REFERENCES
Conway, E. J. (1950). Microdiffusion Analyysis and Volumetric
Error, 3rd ed. p. 241. London: Crosby Lockwood
and Son Ltd.
Friedemann, T. E., Cotonio, M. & Shaffer, P. A. (1927).
J. biol. Chem. 73, 335.
Friedemann, T. E. & Graeser, J. B. (1933). J. biol. Chem.
100, 291.
Friedemann, T. E. & Kendall, A. I. (1929). J. biol. Chem. 82,
23.
Gordon, J. J. & Quastel, J. H. (1939). Biochem. J. 33, 1332.
Long, C. (1946). Biochem. J. 40, 27.
Markham, R. (1942). Biochem. J. 36, 790.
Neuberg, C., Strauss, E. & Lipkin, L. E. (1944). Arch.
Biochem. 4, 101.
Nossal, P. M. (1951). Biochem. J. 50, 349.
Winnick, T. (1942). J. biol. Chem. 142, 451.
Active Transport of Sodium Ions from the Yeast
Cell
BY E. J. CONWAY, H. RYAN AND ENID CARTON
Department of Biochemi8try, Univer8ity College, Dublin
(Received 12 November 1953)
It has been shown (Conway & Hingerty, 1948) that
sodium ions which had entered muscle extensively
in rats on a potassium-free diet were actively
extruded on changing the animals to a potassiumrich
diet. The mean half period of extrusion, even
with the plasma potassium above the normal level
within 24 hr. after the change, was about 3 days.
Very recently, Desmedt (1953) has described a more
rapid net excretion of sodium ions from the isolated
frog sartorius in which sodium ions had entered
extensively after immersion in a potassium-free
Ringer solution in the manner described by
Steinbach (1951). To demonstrate the excretion,
companion muscles which were similarly treated

A RAPID COLORIMETRIC METHOD FOR THE DETERMINATION

OF LACTIC ACID IN MILK
BURDET IIEINEMANN
Chief Chemist, Producers Creamery Company, Springfield, Mo.
~[any methods for determining lactic acid in biological fluids have been
reported. Several have been applied successfully to dairy products. Most
of these methods, however, are too time consuming to be used regularly in
the control laboratory. A search was made, therefore, for a method which
was both rapid and reliable: A method using a simple procedure for the
removal of interfering substances; a simple oxidation procedure; and a
simple means of estimating the quantity of lactic acid present.
The method Mendel and Goldscbeider (5) used for determining the lactic
acid in blood seemed to be the most promising, but when applied to milk
gave unreliable results. Many variations were tried, one of which is reported
below. The method for removing interfering substances is almost
identical with that described by Troy and Sharp (7). Their work on precipitation
procedures was repeated to determine the most satisfactory technique
to be used in conjunction with the eolorimetric method, but no change
was found necesssary.
PROCEDURE
Weigh 5 grams of milk in a 50 ml. volumetric flask. Add 30 ml. of
cold water and mix. Dilute a portion of a 25 per cent solution of copper
sulphate 1 : 3 and add dropwise with agitation 2-8 drops of the dilute solution
to the water and milk mixture until precipitation occurs. Warm the
flask in hot water until the contents reach 45 ° C. Add 6 ml. of 25 per cent
copper sulphate solution and mix thoroughly by rotation. Hold the contents
of the flask at 45 ° to 47 ° C. for 8 to 10 minutes. Next loosen any curd
particles which may adhere to the flask with a bent wire. Add 6 ml. of calcium
hydroxide suspension. This suspension is prepared by slaking 300
grams of the best quality calcium oxide using 1400 ml. water and working
through a 24 mesh screen after 30 minutes standing. Before mixing the
lime with the other constituents in the flask, water should be added to bring
the contents to exactly 50 ml. Then mix until the contents are homogeneous
and allow to stand at 45 ° to 47 ° c. for 10 minutes. Cool to 25 ° C. and filter,
discarding the first few ml. of filtrate.
Place 1.0 ml. of the filtrate in a thoroughly clean and dry test tube.
Hold the test tube in ice water and allow 5.0 mI. of cold concentrated sulphuric
acid (arsenic free) to run down the side wall of the test tube. Mix
gently while cooling. Place mixture in boiling water for exactly 5 minutes~
Received for publication March 28, I940.
969
970 BURDET HEINEMANN
Cool in ice water for three. Add 4 drops of a 0.1 per cent solution of veratrole
in water and mix. Hold in ice water for 60 minutes and compare colors with
artificial or natural standards.
Natural standards may be prepared according to Hil!ig (3). Dissolve
106.6 rag. of purified lithium lactate in 100 ml. of distilled water. 1.0 ml.
of this solution contains the equivalent of 0.1 per cent lactic acid. Consequently,
to prepare a 0.01 lactic acid standard, 5.00 ml. of this solution is
diluted to exactly 50.0 ml. and mixed. One ml. of this dilution is then
placed in a test tube and 5 ml. of concentrated sulphuri¢ acid added. The
mixture is held in boiling water 5 minutes and cooled in ice water 3 minutes.
Four drops of a 0.1 per cent solution of veratrole is then added and the color
observed after 60 minutes. Additional standards may be made by diluting
the original lithium lactate solution as required.
Artificial standards which are fairly satisfactory may be prepared according
to the method of Nordb5 (6). Mix 25.0 ml. of a 0.01 per cent solution
of fuchsin in water and 4.75 ml. of a 0.01 per cent solution of Tropaolin
000. (The mixture must be diluted immediately for a precipitate forms on
standing.) One tenth ml. of this mixture diluted to 50.0 ml. of solution is
equivalent to approximately 0.005 per cent lactic acid; 0.25 ml. diluted to 50
ml. is equivalent to approximately 0.01 per cent and ~ 0.9 ml. diluted to 50
ml. is equivalent to 0.02 per cent. NordbS's standards are only suitable for
concentrations below 0.03 per cent. Above this percentage, the yellow color
increases in intensity. Consequently, for a 0.05 per cent lactic acid standard,
it is necessary to add 0.4 ml. of .01 per cent Tropaolin and 1.8 ml. of
.01 per cent fuchsin to 47.8 ml. of water; for a 0.07 per cent standard, 1.0
ml. of Tropaolin and 2.0 ml. of fuchsin to 47.0 ml. of water; and for a 0.10
per cent standard, 2.0 ml. of Tropaolin and 2.0 ml. of fuchsin to 46.0 ml. of
water. These artificial standards should be checked against natural standards
before using.
DISCUSSION
According to the authors, the original test which was applied to blood is
accurate to -+- 0.004 per cent between 0.001 and 0.05 per cent (5) and 1 part
in 400,000 lactic acid could be detected. Mendel (4) later made two improvements
which he stated made possible the detection of one part in
1,000,000. I-Iowever, when applied to milk, the original test was sensitive
to 0.01 per cent and had a limited degree of accuracy (± 0.04 per cent).
With the modification outlined above, the test is sensitive to 0.005 per cent
and accurate to ± 0.004 per cent between the ranges Of 0.005 and 0.12 per
cent lactic acid providing the procedure is closely followed.
Mendel (4) has pointed out that small changes in the concentration of
the sulphuric acid may lead to inaccurate results. A difference of 2 to 3
per cent in the concentration may result in as much as a 15 to 20 per cent

DETERMINING LACTIC ACID IN MILK 971

error. It is wise, therefore, to check frequently the color of the artificial
standards. In this respect, it should be noted that the individual analyst
should try several different quantities of sulphuric acid (i.e., 4.5, 4.75, 5.0
ml. etc.,) in the preparation of natural standards in order to ascertain the
exact amount to add to the 1.0 ml. filtrate to obtain the greatest degree of
color development for each batch of sulphuric acid. Derviz (1) describes a
method for purifying and storing sulphuric acid so as to maintain a constant
concentration.
1Vfendel (4) makes the recommendation that after the mixture of acid and
filtrate has been held in boiling water five minutes, it be cooled 2 minutes in
ice water, veratrole added, and set at 25 ° C. After 20 minutes, the color
may be compared with standards similarly treated. This method may be
employed whenever results of great accuracy are not desired. ~
Mendel and Goldscheider (5) used 0.1 ml. of a 0.125 per cent veratrolc
in 99.8 per cent alcohol. Other investigators suggested 0.05 ml. of a 1 : 800
solution in 96 per cent alcohol (6), 0.1 ml. of 20 per cent veratrole solution
in glacial acetic acid (1) and 0.1 ml. of 20 per cent in absolute alcohol (2).
These were all tried, but none gave as satisfactory results as 4 drops of a
0.1 per cent solution in water. Various solutions of guaicol, hydroquinone,
and Schiffs reagent were also tried and considered unsatisfactory.
Although the original method called for a 4 minute heating period, 0.5
ml. filtrate and 3.0 ml. of conc. tt2SO, it was found that 5 minute heating
period, 1.0 ml. filtrate and 5.0 ml. of concentrated sulphuric acid gave more
uniform results.
The same substances interfere with this method as those Troy and Sharp
(7) found to interfere with theirs. Formaldehyde, overneutralization,
rancidity, sucrose, and products resulting from the heating of milk at or
near the boiling point for one half hour or longer resulted in high values.
Mendel and Goldscheider state, however, that acetone, ~-oxybutyric acid,
acetic acid, urea, uric acid, creatin, creatinin, glycocol, alanine, and propionic
acid, do not interfere with the color development.
Traces of organic matter interfere with proper color development and
consequently the test tubes must be thoroughly clean and kept stoppered
as much as possible during the determination.
When greater rapidity of testing is required, the precipitate of proteins,
lactose and fat, may be centrifuged. In this case, cream test bottles (18
gram body, 9 gram neck, graduated in 1 per cent between 0 and 55 per cent)
were selected from stock and graduated to contain 50.0 ml. at 20 ° C. The
milk was weighed in these bottles which, after precipitation and cooling,
were placed in a Babcock tester and centrifuged 4 minutes. The centrifugate
was then decanted through a fluted filter, the first few ml. of filtrate
being discarded.

972 BURDET HEINEMANN

SUMMARY
A rapid colorimetric method for the quantitative estimation of lactic
acid in milk is described. The steps involved are precipitation of fat, lactose
and protein with copper sulphate and calcium hydroxide, filtering,
adding concentrated sulphuric acid (arsenic free) to the filtrate, heating,
cooling, and adding veratrole. A red color develops on standing which is in
proportion to the amount of lactic acid present.
The test is sensitive to about 0.005 per cent and is accurate to ___ 0.004
per cent between the ranges of 0.005 and 0.12 per cent lactic acid.
:REFERENCES
(1) DERWZ, G.V. The colorimetric lactic acid determination according to Mendel-Goldscheider.
Zhur. Exptl. Biol. Med. 12: 147-50. 1929. (Original not seen.
See Chem. Abstr. 24- 1880. 1930.)
(2) FUCHS, HANS J. Einige Verbesserungen der kolorimetrischen Milchsfiurcbestimmung
nach Mendel und Goldscheider. Biochem. Ztschr. 217: 405-8. 1930.
(3) HILLIG, FRED. The colorimctric determination of lactic acid in milk and milk products.
J.A.O.A.C. 20: 130-40. 1937.
(4) MENDEL, BRUNO. Zur Methode der k01orimetrischen Milchs~urebestimmnng. Biochem.
Ztschr. 202: 390. 1928.
(5) MENDEL, BRUNO AND GOLDSCYIEIDER, INGEBORG. Eine kolorimetrische Mikromethode
zur quantitativen Bestimmung der Milchsiiure im Blut. Biochem. Ztschr. 164:
163-74. 1925.
(6) NORDBh, I:~AGNVALD. Zur Methode der Milchs~urebestimmung nach Mendel und Goldscheider.
Biochem. Ztschr. 271: 213-215. ]934.
(7) TROY, It. C. AND SHARP, P. F. Quantitative determination of lactic acid in dairy
products. Corner Univ. Ag. Exp. Sta. Memoir 179. 1935.