GASOMETRIC DETERMINATION OF METHEMOGLOBIN.

BY DONALD D. VAN SLYKE AND ALMA HILLER.

(FTom the Hospital o-f The Rockefeller Institute for ,Xedical Research,
New York.)

(Received for publication, July 10, 1929.)

In the present paper the method of Van Slyke (1) for methemo-
globin determination has been modified by employing technique

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developed by the writers (2) for determining the carbon monoxide-
binding capacity of blood. The principle, as in the former methe-
moglobin method (l), is that introduced by Nicloux and Fontes (3).
Two determinations are required. ,In one (A) the normal or active
form of hemoglobin, capable of binding 02 and CO, is determined
by measuring the CO-binding capacity of the hemoglobin-methe-
moglobin mixture. In the other (B) sodium hydrosulfite is added,
changing methemoglobin into active reduced hemoglobin, and the
total hemoglobin is determined by the CO-binding capacity. The
difference, B - A, indicates the methemoglobin. The technique
here introduced has the advantage that all the operations, reduc-
tion with hydrosulfite, saturation with CO, and determination of
CO bound by hemoglobin, are carried out in the chamber of the
Van Slyke-Neil1 apparatus (4, 5). In consequence, the procedure
is more simple and rapid than that previously presented (l), and
requires much less blood, as little as 0.2 cc., or even 0.1 cc., suffic-
ing for an analysis.
Conant, Scott, and Douglass (6) have shown that the hydro-
sulfite-CO procedure is not applicable in the presence of hematin,
because the latter is reduced and behaves like methemoglobin in
binding CO. They use titanous tartrate to reduce the methemo-
globin, and determine the Or-, instead of the CO-, binding capacity.
Their procedure is not affected by the presence of hematin. The
occurrence of hematin in blood is rare, however, except under ex-
perimental conditions designed to produce it. We have accord-
ingly for simplicity retained the hydrosulfite-carbon monoxide
procedure.
205
 206 Gasometric Methemoglobin

In determining the carbon monoxide capacity in blood reduced

by hydrosulfite, the only modification necessary to our former
procedure (2) for hemoglobin determination is the use of higher CO
tensions to saturate the hemoglobin. The presence of hydro-
sulfite and ammonia appears to lower somewhat the affinity of
reduced hemoglobin for CO, so that 100 mm. tension of the latter,
instead of only 30, are required to insure complete conversion of
the hemoglobin to carboxyhemoglobin.
Reagents.

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Nzclouxs ammoniacal sodium hydrosulfite solution is prepared as
previously described (1). The carbon monoxide gas, 1 N air-free
sodium hydroxide, and 6 N sodium hydroxide are prepared and
handled as outlined by Van Slyke and Hiller (2).
The acid ferricyanide solution is prepared as follows, with more
acid than formerly. To 92 volumes of a stock solution containing
32 gm. of &Fe(CN)G per 100 cc. are added 20 volumes of con-
centrated lactic acid, of specific gravity 1.2. The ferricyanide in
this acidified solution undergoes slow decomposition, but if kept
out of direct sunlight can be used for about 2 months.
Procedure.
Determination of Active Hemoglobin.-The carbon monoxide
capacity method of Van Slyke and Hiller (2) is used without
change.
Determination of Total Hemoglobin. For 2 Cc. of Blood.-2
drops of caprylic alcohol are drawn into the capillary beneath the
cup of the manometric apparatus. Into the cup are measured
4.3 cc. of water. With a stop-cock pipette provided with a rubber
tip (see Fig. 4, p. 532, of Van Slyke and Neil1 (4) ), 2 cc. of blood are
run directly into the chamber, followed by a few drops of the
water in the cup to wash the blood through the capillary. From
a micro burettei 0.4 cc. of the ammoniacal sodium hydrosulfite
solution is run into the chamber, followed by the remaining water
in the cup. 1 or 2 cc. of mercury are placed in the cup above the

1 The micro burette used for measuring the hydrosulfite and the acid
ferricyanide was made by sealing a stop-cock onto a pipette graduated in
0.01 cc. divisions. The delivery capillary was provided with a rubber tip
as shown in Fig. 3, p. 125 of Van Slykes paper (5) and described on p. 126.
 D. D. Van Slyke and A. Hiller 207

chamber. Carbon monoxide sufficient to give 150 mm. of pres-

sure is measured into the chamber from a modified Hempel pipette
in t,he manner described on pp. 816 and 817 of our previous paper
(2).
The equilibration of the blood solution with CO is carried out as
described on p. 811 of our former paper (2), except that a little
more time seems necessary. We shake the chamber 1.5 instead
of 1.0 minute.
The determination of CO bound as HbCO is also carried out in all

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details, including the c correction, as described on the same page,
except that 0.3 cc. instead of 0.25 cc. of acid ferricyanide solution
is added. The value of c in the present procedure is somewhat
greater than in the carbon monoxide capacity method (a), because
of the greater amount of CO physically dissolved by the blood
solut,ion at the higher CO pressure used for saturation. The
value of c which we find in our laboratory, with a temperature of
20-25 is about 14.0 mm. Each analyst should, however, deter-
mine it for himself repeatedly.
The cleaning of the chamber after each analysis is more im-
portant in this analysis than in the simple carbon monoxide capac-
ity determination, because in the present case any particle of
methemoglobin ferricyanide precipitate left adhering to the walls
of the chamber will be reduced in the next determination to active
hemoglobin by hydrosulfite, and added to the total hemoglobin
found. A little of the hydrosulfite solution added to the first
portion of water used to clean the apparatus assists in dissolving
such particles quickly. The procedure for rapid and complete
washing of the apparatus is described on p. 813 of our former
paper (2).
For 1 Cc. Blood Samples.-The procedure is the same as that
used for 2 cc. samples except that half as great a volume of each
reagent is used for 1 cc. of blood as for 2 cc. The pressure of CO
used is the same, 150 mm.
Micro Determination of Total Hemoglobin with 0.1 CC. or 0.2 Cc.
Blood Samples.-The procedure for measuring the blood and
transferring it to the chamber of the apparatus is the same as that
described on p. 814 of our previous paper (2), except that 0.05 cc.
of the ammoniacal sodium hydrosulfite solution is run into the
chamber of t,he apparatus before the final washing of the cup SO
208 Gasometric Methemoglobin

that this volume of fluid is part of the total 2 cc. measured into the
chamber. Carbon monoxide to 150 mm. pressure is admitted into
the evacuated chamber in the same manner described above.
Of the acid ferricyanide solution only 0.05 cc. is added. It is run
in while the blood solution is still in the top of the chamber and is
followed by several drops of mercury which break up the methemo-
globin precipitate into fine particles. The procedure is continued
as detailed on p. 815 of our former paper (2).
The c correction for the micro method is somewhat over 20 mm.

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It must be redetermined with each set of micro analyses.
The calculations for total hemoglobin are the same as those de-
scribed in the former paper (2) on pp. 812 and 813.
CO capacity = (pl - p, - c) X f.

For 2 cc. blood samples f is a factor from the last column of

Table II or III of Van Slyke and Neil1 (4). For 1 cc. samples f
is found in the seventh column of their Table II or III, when S is
3.5 cc. and a is 2.0 cc., or in the sixth column when the final gas
pressure is read with the gas at 0.5 cc. volume. When 0.2 cc. of
blood is employed f is found in the fifth column of their Table II
or III. When the sample is 0.1 cc. the factors used for 0.2 cc. are
multiplied by 2.
Methemoglobin = total hemoglobin - active hemoglobm.

EXPERIMENTAL.

Tension of CO Required for Complete Conversion of Reduced Hb

into HbCO.-The material used was blood in which part of the
hemoglobin had been oxidized to methemoglobin by addition of
measured amounts of ferricyanide. The amount of ferricyanidc
taken was 0.5 mol per mol of hemoglobin present (1 mol of Hb
being estimated as the amount binding 1 mol of O2 or CO). As
would be predicted from the results of Conant and Fieser (7)
this procedure produces a mixture of approximately equal parts
of active hemoglobin and methemoglobin.
To 50 cc. of blood with 18.41 volumes per cent of CO-binding
capacity Mercks saponin was added with shaking until hemolysis
was complete, then 0.3 cc. of a solution containing 32 gm. of
potassium ferricyanide per 100 cc. was added. 2 cc. portions of
 D. D. Van Slyke and A. Hiller
TABLE I.
Tension of CO Required for Quantitative Saturation of Blood for Total
Hemoglobin Estimation.
Tension of CO at
CO capacity of blood beginning CO bound per 100 CD. of treated blood.
before oxidation. of saturation.
, ,
per cent CO bound by
rd. per cent ,Il?7L. cc. untreated blood
I 1
18.41 35 17.25 93.7
70 18.23 99.0
100 18.50 100.5
150 18.44 100.2

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--Macro and Micro Determinations
TABLE II.

of the Methemoglobin Content of Blood.

I 1
Total Unoxidized
Quantity of CO capacity Methemoglobin
hemoglobin in hemoglobin in
blood used for of blood before in terms of
terms of co terms of co
smlysis. oxidation. CO capacity.
capacity. capacity.

cc. vol. per cent cd. per cent vol. per cent
2 18.41 18.34 9.01
18.40 18.51 8.93
18.47
_-
Average.. .. . 18.41 18.44 8.97 9.47

I 18.32 8.82
18.38 8.96
18.46
_
Average.. .. . 18.39 8.89 9.50
_
0.2 18.40 8.76
18.15 8.83
18.51

Average.. ... 18.35 8.80 9.55

0.1 18.67 8.53

17.81 8.76
18.35

Average.. ... 18.28 9.63

i

this blood were analyzed for total hemoglobin as described above,

by treatment with hydrosulfite and CO. The procedure was
varied: however, with respect to the CO tensions used to saturate
210 Gasometric Methemoglobin

the blood in the chamber. The results, given in Table I, show

that 100 mm. of CO tension are adequate.
Constancy of Results.-The same ox blood was partially oxidized
with ferricyanide in the same manner. The mixture was imme-
diately analyzed for total hemoglobin and for active hemoglobin.
Determinations were carried out with 2 cc., 1 cc., 0.2 cc., and 0.1
cc. samples. The results recorded in Table II indicate the order of
constancy obtained by the method.
SUMMARY.

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The gasometric method for the determination of methemo-
globin has been simplified by adapting to it the carbon monoxide
capacity technique of the authors. The entire procedure is
carried out in the manometric apparatus of Van Slyke and Neill.
Amounts of blood varying from 2 cc. to 0.1 cc. can be used for the
analyses.
BIBLIOGRAPHY.

1. Van Slyke, D. D., J. Biol. Chem., 66,409 (1925).

2. Van Slyke, D. D., and Hiller, A., J. Biol. Chem., 78,807 (1928).
3. Nicloux, M., and Fontes, G., Bull. Sot. chim. biol i 6.725 (1924).
4. Van Slyke, D. D., and Neil& J. M., J. Biol. Chem., 61,523 (1924).
5. Van Slyke, D. D., J. Biol. Chem., 73,121 (1927).
6. Conant, J. B., Scott, N. D., and Douglass, W. F., J. Biol. Chem., 76,
223 (1928).
7. Conant, J. B., andFieser, L. F., J. Biol. Chem., 62,595, 623 (1924-25).
GASOMETRIC DETERMINATION OF
METHEMOGLOBIN
Donald D. Van Slyke and Alma Hiller
J. Biol. Chem. 1929, 84:205-210.

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