Notes on Methemoglobin
I
S
Tnomas Leany
HE AUTHORS
Evelyn
and
were recently
Malloy
method
*
Determination
I
ana Roger Smit
made aware
that a modification
for the determination
of the
of methemoglobth
in blood (1) is needed before this procedure can be used effectively
with a Coleman Jr. spectrophotometer.
The necessity for this modification will be demonstrated
from the point of view of instrumental
error later in this paper.
In general, specimens submitted to this laboratory
for methemoglobin determinations
fall into one or the other of two classes: (1)
those in which an acute exposure to a methemoglobin-forming
agent
was suspected, and (2) those in which the occupational
nature of the
subject involved chronic contact with such agents or suspected
agents.
as part
In the latter
of a periodic
class, the methemoglobin
physical
examination.
was routinely
requested
The limits of precision of the Evelyn-Malloy
method have been
placed at 0.1 to 0.2 gm./100 ml. of blood (2), corresponding
to 0.66 to
1.33 per cent of the total pigment.t
The clinical manifestations
of
cyanosis, on the other hand, have been reported to occur when the
methemoglobin
level reaches about 3 gm./100 ml., or 20 per cent of
the total pigment
(3). It is therefore
obvious, that considerable
leeway is left for methemoglobin
concentrations
in subclinical levels.
By decreasing the effective instrument
error and possibly increasing the sensitivity by using a lower dilution of blood (4), we propose
that it may be possible to evaluate subclinical levels of methemoglobin
in_patients
occupationally
exposed to agents capable of elevating it
From the Chemistry Department of the USAREUB
Medical Laboratory,
APO 180, New
York, N. Y.
*preeent address:9925 So. Seeley Ave., Chicago, Ill.
tFor comparative purposes, the percentage of the total pigment indicated here and in the
following text have been computed from the given methemoglobin concentration in gni./100
ml. of blood on the basis of 15 gm./100 nil, as an approximate over-all mean for the total
hemoglobin in men and women.
Received for publication
Sept. 15, 1959.
148
Vol. 6, No. 2, 1960
149
METHEMOGLOBIN DETERMINATION
above the normal. Such a proposal would not have been feasible on
a Coleman Jr. spectrophotometer
without our modification
of the
original Evelyn-Malloy
method. Direct expression of the final results
as per cent methemoglobin
of the total blood pigment might be another advantageous
modification.
MATERIALSAND METHODS
The reagents of Evelyn and Malloy (1) were used throughout.
The
instrument
utilized was a Coleman Jr. Model 6A spectrophotometer.
The procedure
is as follows. To four standard
19- X 125-mm.
cuvets labelled A, B, C, and D, add 10, 9.7, 7, and 7 ml. of M/60 phosphate buffer respectively.
To cuvet B, add 0.3 ml. of capillary
or
oxalated blood. Mix well and allow the tubes to stand for 5 minutes.
Read the optical density of Tube B at 635 m, using Tube A to set
zero. This reading is D1. Add to Tube A and to Tube B, 2 drops of
neutralized
sodium cyanide and mix well. Allow the tubes to stand
for 2 minutes and read as before. This reading is D2. Add to Tube
A and to Tube B, 0.1 ml. of 10% potassium
ferricyanide.
Mix well,
and transfer
1 ml. from each of Tubes A and B to Tubes C and D,
respectively.
Mix well, and read Tube D at 540 m, using Tube C to
set zero. This reading is P3. The calculations
are then:
% Metllb
of the total pigment
X K
=
D3
The method is standardized
as follows. Set up two cuvets as A and
B in the procedure, and add the blood to Tube B. Add ferricyanide
to both tubes and mix as before. After allowing the mixture to stand
for 5 minutes, read Tube B against Tube A at 635 m. This reading
is D4. Add two drops of neutralized
sodium cyanide to each tube, and
mix the contents thoroughly.
Allow the tubes to stand a few minutes
and again read Tube B against Tube A. This reading is D5. Determine the hemoglobin concentration
of the blood sample by the method
of Drabkin (5, 6). The constant is then calculated as:
K
=
gm. Hb/100 ml. blood
<
D4-D5
The K value as determined
for this laboratory
was 63.5.
RESULTS
An examination
of a graph of effective instrument
error versus
percentage
transmission
for the Coleman Jr. spectrophotometer
150
LEAHY & SMITH
Clinical Chemhtry
shows that at the extreme ranges of % T, the instrument
error rises
quite sharply (7). Although the manufacturer
recommends
limiting
readings to the 5 to 90 % T range, considerable
instrument
error is
still possible within these limits. We have, therefore, chosen our concentrations
so that the readings fall into the 5 to 70 % T range. The
maximum effective instrument
error is then limited to 3 per cent
instead of a possible 10 per cent.
Table 1 illustrates
the differences in instrument
error between the
original Evelyn-Malloy
method and our method as used with a Coleman Jr. instrument.
It can be seen from Table 1 that the effective
instrument
error has been reduced sharply in the crucial D1 and D2
readings to approximately
the lowest possible effective error of the
instrument.
The foregoing procedure was then utilized to determine the methemoglobin content in blood specimens from 18 normal young men
who received prior screening interviews with emphasis on the taking
of drugs, engaging in painting, or exposure to petroleum fuels. The
mean value obtained on the 18 single samples was 0.68 per cent methemoglobin of the total pigment, with a standard
deviation
of 0.5.
The range was from 0 to 2.0 per cent of the total pigment. The rough
criterion of ± 3 standard deviation as including 99 per cent of a population would give a range of 0 to 2.2 per cent.
Our modified procedure was then applied to specimens drawn from
55 persons from the following three occupational
groups:
spray
painters, fuel workers, and processors.
Processors
differ from fuel
workers in that the former handle closed containers
of petroleum
fuels rather than being involved in actual fueling operations.
The
results obtained are indicated in Table 2. It should be noted that
although the mean for the methemoglobin
level in processors
(petroTable
1. Epy’xc’rivx
D1
D2
D3
D4
D5
T Range for
E-M method
85-92
85-92
falling
R.
Inst.
ercor
6.5-11
6.5-11
Not used
40-50
75-85
For specimens
trations.
ON A COLEMAN
METHOD COMPARED WITH
%
Reading
Enuous
INSTRUMENT
ORIGINAL EVELYN-MALLOY
Not used
2
4-6.5
within
the normal
%
Ja.
THE
Ton THE
SPECTROPHOTOMETER
MODIJ’IED METHOD Dancamsm
T Range for
our method
Efi. Inst.
.rroi
Reductson
ia error
55-70
2-3
359*
55-70
2-3
3.5-9
25-40
8-15
35-50
2
2-3
2
-(0-1)
2-4.5
or subclinical
range
of methemoglobin
concen-
Vol. 6, No. 2, 1960
Table 2. RELanvz
Mws
OF TUE
PERCENTAGE
POE 55 PERsONs ENGAGED
Occupation
No.
Processors
151
METHEMOGLOBIN DETERMINATION
of
IN
OF
Thana
DIFFERENT
Mean
case,
OF THE TOTAL
METHEMOGLOBIN
%
PIGMENT
OCCUPATIONS
MetHb
Rtandard
deviation
45
1.17
0.46
Fuel workers
6
1.43
0.36*
Spray
4
1.55
0.33*
painters
*]c1uded
computation
only for comparative
purposes
of no statistical
consequence.
since
the small
number
of cases
renders
the
leum products
depot workers)
falls within the proposed
normal
range, a statistically
significant difference exists between the mean
of this group and the mean of the normal group at a 0.1 per cent significance level when using a t distribution
(8).
Often, when a clinical method is made more sensitive, it must be
applied and interpreted
with a greater degree of caution. It was our
experience that during a routine survey of personnel employed in a
petroleum
products testing laboratory,
only two specimens showed
values above 1 per cent of the total pigment.
Herr A, a chemist,
showed 1.6 per cent while Frau B, whose duties consisted of washing
glassware, showed 1.5 per cent. Because these two samples were high
in relation to the rest of the group and since there was no apparent
explanation,
new samples were drawn after 3 weeks. The results of
these samples were 3.9 per cent for Herr A and 2.3 per cent for Frau
B. During a subsequent interview with these subjects, it was revealed
that both had been taking medication
prior to the first survey and
continuing
through
the recheck.
Herr A was taking Myocardin
(Byk-Gulden-Lomberg,
Konstanz),
a combination
of nitroglycerin,
atropine methyl nitrate, phenobarbital,
papaverine,
and euphylline
for a coronary condition, while Frau B was taking Melabon (Dr.
Rentschler
& Co., Laupheim i. W#{252}rtt),
an antipyrine,
aminopyrine,
phenacetin, isopropyl phenazon combination for migraine attacks.
SUMMARY
The Evelyn-Malloy
procedure for the determination
of methemoglobin in blood has been modified for use with a Coleman Jr. spectrophotometer
by using a lower dilution of blood to reduce the effective
instrument
error from a possible 10 per cent to a possible 3 per cent.
The method has been further modified to give results directly as
the per cent methemoglobin
of the total blood pigment.
152
LEAHY & SMITH
Clinical Ch.misfry
The normal range for this determination
appears to be up to 2 per
cent of the total pigment present as methemoglobin.
A statistically
significant
difference
was observed between the
means of the methemoglobin
percentages
of a normal group and a
group of persons whose occupation placed them in frequent contact
with petroleum fuels.
REFERENCES
1. Evelyn,
K. A., and Malloy,
2.
P. B.,
Hawk,
4.
5.
6.
7.
8.
Practical
Physi.ologico2
Chetni8try,
Ed.,
Cantarow, A., and Trumper, M., Clinical
Biochemistry,
5th Ed., Philadelphia,
W. B.
Saunders, 1955, p. 208.
Paul, W. D., and Kemp, C. B., Proc. Soc. Exptl. Biol. 4 Med. 56, 55 (1954).
Drabkin, D. L., Amer. J. Med. Sci. 215, 110 (1948).
Drabkin, D. L., Amer. 1. Med. Sci. 217, 710 (1949).
Operating Directions for the Model 6A Coleman Junior Spectrophotometer,
Coleman
Instruments,
Inc., Maywood, fll., 1945, p. 40.
Dixon, W. 3., and Massey, F. J., Introduction
to Statistical
Analysis,
Sad Ed., New
York, McGraw-Hill, 1957.
18th
3.
H. T., J. Biol. Chem. 126, 655 (1938).
Oser, B. L., and Sumnierson,
W. B.,
New York, Blakiston,
1954, p. 621.