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Abstract ]
A method for the determination of methemoglobin in the
presence of other hemoglobin subforms (i.e., oxy-, deoxy-, and
Experimental
Apparatus
Introduction
Methemoglobinemia is a pathological condition in which
iron in hemoglobin is in its tervalent oxidation state (Fe3
rather than its divalent one (Fe2 this results in the formation
of a hemoglobin subform that is unfit for transporting oxygen.
Methemoglobinin blood can originate from in vivo exposure to
oxidants (1-3), which gives rise to a variably serious picture of
tissue hypoxia, from heating of blood (e.g., in charred bodies)
(4) or even from specific storage conditions (e.g., refrigeration, freezing) (5-8).
In any case, determinations of methemoglobin are relatively
commonplace and useful to toxicological laboratories, so a
need exists for simple and reliable analytical methods for the
identification and quantitation of this hemoglobin subform in
blood. A large number of methods for this purpose have so far
been reported, the most common of which involve spectrophotometric multicomponent analysis (9-11).
This paper reports a method for the determination of methemoglobin in the presence of the hemoglobin subforms most fre-
Samples
The samples studied consisted of fresh blood collected in
Venojecttubes (Terumo)containing EDTAas anticoagulant. All
were obtained from the BiochemistryLaboratoryof the Galician
General Hospital.
Reagents
The reagents used included pure oxygen, carbon monoxide,
and nitrogen (all obtained from Air Liquide);a 13% hemoglobin
standard supplied by the Biochemistry Laboratory of the Galician General Hospital; a 5-mg/dL standard of bilirubin from
Bayer; potassium ferricyanide [K3Fe(CN)6],sodium dithionite
(Na2S204), crystalline sodium nitrite (NaNO3),Methylene Blue
for microscopy (16316), 37% hydrochloric acid, and sodium
hydroxide from Merck.
67
moglobin in the presence of other hemoglobin subforms. Finally, the quantitative study in the first-derivative mode (D1) ,
between 700 and 500 nm, was done.
Methods
Preparationof standards
Oxyhemoglobin(OxyHb),deoxyhemoglobin(DeoxyHb),carboxyhemoglobin (COHb), and methemoglobin (MetHb) standards were prepared from a stock standard containing 13 g% of
hemoglobin. Followingdilution to 1% (v/v) in distilled water, a
100% saturated solution of oxyhemoglobin was obtained by
bubbling oxygen for 10 min, excess O2 being removed by bubbling N2for 5 rain. The resulting solution was used to make the
following four aliquots: aliquot A, which consisted of oxyhemoglobin; aliquot B (deoxyhemoglobin),which was obtained by
adding sodium nitrite; aliquot C (carboxyhemoglobin),which
was prepared by bubbling CO for 5 rain, followedby N2 to remove excess gas from the solution; and aliquot D (methemoglobin), which was obtained by oversaturation with
potassium ferricyanide.
Recordingof spectra
Absorbance and derivative spectra (D1, D2, and D3) for the
pure standards were recorded in order to determine the best
spectrophotometric conditions for the determination of methe-
[Hb] in the
solution
(mg/mL)
Vol Standard A
(100% MetHb)
(mL)
Vol StandardB
(100% OxyHb)
(mL)
1.3
1.3
1.3
1.3
1.3
1.3,
1.3
1.3
1.3
1.3
1.3
0.0
0.2
0.4
0.6
0.8
1
2
4
6
8
10
10
9.8
9.6
9.4
9.2
9
8
6
4
2
0
MetHb
%
100
100
100
100
100
I00
100
100
100
68
[Hb]
in the
sample
(g/dL)
2.6
5.2
7.8
10.4
13.0
15.6
18.2
20.8
23.4
[Hb] in the
solution(after
Vol.
1% dilution) StandardC
(mg/mt)
(mL)
0.26
0.52
0.78
1.04
1.30
1.56
1.82
2.08
2.34
1
2
3
4
5
6
7
8
9
Vol.
Distilled
water
(mL)
9
8
7
6
5
4
3
2
1
Procedures
Determination of the methemoglobin saturation percentage.
Following dilution of the 13 g% hemoglobin (Hb) standard to
1% in distilledwater and 100% saturation with oxygen, the followingtwo working standards were prepared: standard A, which
contained 1.3 mg/mL MetHb (followingsaturation with potassium ferricyanide)and standard B, which contained 1.3 mg/mL
OxyHb.
Mixtures of the previous two standards in appropriate proportions gave the solutions listed in TableI, which contained increasing amounts of MetHb but the same concentration ofHb.
Determination of the total hemoglobin concentration. The 13
g% hemoglobinstandard was used to prepare another standard,
C, containing2.6 mg/mL methemoglobin(i.e., 100% MetHb).By
appropriate dilution, the solutions listed in TableII, Whichcontained variable concentrations of MetHb,were prepared.
Study of interferences
The interferences examined were those of the other
hemoglobin subforms, potassium ferricyanide, other plasma
components, and even a therapeutic agent used to treat methemoglobinemia (MethyleneBlue).
4
6
8
10
20
40
60
80
100
1D64snm
SD
(n = 5)
CV(%)
(n = 5)
0.0500
0.1229
0.1551
0.1999
0.2641
0.3252
0.5603
1.0753
1.5448
2.0574
2.5710
0.003
0.004
0.010
0.006
0.013
0.0t0
0.013
0.005
0.022
0.012
0.001
5.15
3.44
6.41
2.75
4.86
3.10
2.35
0.43
1.42
0.60
0.05
4,MO
Z,4000
l,.f~lm
8.nnan
o,gM
c~
8.5510
"4,8000
6,44N
-Z,4000
O.~ii
9,4,NOt t
~,O
O,MM
Qi.I
m.O
~,0
M,O
nm
rim
'"
5 . i
"1
II.U
t 6 z'm
"ll.U
0.9609
-2.4El
4,~0
-Z.IIN I
SN.II
~.11
.
r.li.O
.
r.,,~,11
780.0
nm
69
~'
Table IV. Quantitation of MetHb in Solutions Containing
Increasing Concentrations of Hemoglobin
(Methemoglobin)
[Hb] in the [Hb] in the
MetHb sample(real) solution
%
g/dL
mg/mL
100
100
100
100
100
100
100
100
1O0
2.6
5.2
7.8
10.4
13.0
15.6
18.2
20.8
23.4
0.26
0.52
0.78
1.04
1.30
1.56
1.82
2.08
2.34
1D64snm
0.5145
1.0552
1.5324
2.0698
2.5704
3.1191
3.6265
4.1623
4.6744
SD
(n = 5)
CV (%)
(n = 5)
0.0096
0.0121
0.0160
0.0217
0.0199
1.87
1.15
1.04
1.05
0.53
0.0232
0.74
0.0292
0.0218
0.80
0.52
0.0207
0.44
[Hb] D1.Method
[Hb] Coulter
1D645nm
(g/dL)
(g/dL)
1
2
3
4
5
1.3472
1.5786
1.9967
1.9580
2.0454
6.7
7.9
9.9
9.8
10.2
6.4
7.6
9.8
10.3
10.5
6
7
8
9
10
1.9533
2.2936
2.4561
2.9429
2.7879
9.7
11.4
12.2
14.7
11.1
12.2
13.6
14.4
11
12
13
14
15
2.8969
2.8549
2.9127
2.9871
3.1909
13.9
14.5
14.2
14.5
14.9
15.9
14.5
14.8
14.8
15.2
15.5
16.2
NO
70
(Eq
18
y = 0.9634X
r = 0.9841
16
.:3:. 8
~10
=/
0/0
6
6.0
8.0
10.0
12.0
14.0
16.0
18.0
Record D1 spectrum
between 700-500 nm
MeasureID64snm ~
Saturatethe
dilutionto 100% ~
with 02
Saturateto 100%
with Potassium
Ferricyanide
Avalue ]
Record DI spectrum
between 700-500 nm
Measure 1D64snm
Interferences
Effect of turbidity. In order to determine the potential influence of turbidity on the determination of methemoglobin,
serum with a high triglycerideconcentration was supplied to diluted blood at increasing concentrations from 0 to 3075 mg/dL.
Interferences were found to be significant above a triglyceride
concentration of 800 mg/dL---equivalent of a high sample
opacity--which caused the percent MetHb saturation to rise to
1%; in fact, a 3000-mg/dL concentration simulated a MetHb
saturation of 5.5%. We should note, however, that normal
triglyceride levels in blood are below 160 mg/dL and that concentrations above 300 mg/dL are definitely pathological.
Conclusions
The proposed spectrophotometric method, based on the first
derivative of the spectrum at 645 nm, allows the determination
of the methemoglobin saturation percentage and the total
hemoglobin concentration in the sample. This affordsbetter interpretation of the analytical results because it allows the absolute amount of functional hemoglobin in an individual to be
determined. Basedon the results, neither the presence of other
hemoglobin subforms nor that of endogenous plasma components (bilirubin and triglycerides) or even therapeutic agents
such as Methylene Blue interferes with the proposed method.
References
1. S.M. Bradbery, R.M. Whittington, D.A. Parry, and J.A. Vale. Fatal
methemoglobinemia due to inhalation of isobutyl nitrite. Clin.
Toxicol. 32(2): 179-184 (1994).
2. P.M. Wax and R.S. Hoffman. Methemoglobinemia: an occupational hazard of phenyl-propanolamine production. Clin. Toxicol. 32(3): 299-303 (1994).
3. C.L. French, S.S. Yaun, L.A. Baldwin, D.A. Leonard, X.Q. Zhao,
and E.J. Calabrese. Potency ranking of methemoglobin-forming
agents. J. Appl. Toxicol. 15(3): 167-174 (1995)
4. G.P. Fechner and D.J. Gee. Study on the effects of heat on blood
and on the post-mortem estimation of carboxyhaemoglobin and
methaemoglobin. Forensic Sci. Int. 40:63-67 (1989).
5. K. Sato, K. Tamaki, H. Okajima, and Y. Katsumata. Long-term
storage of blood samples as whole blood at extremely low temperatures for methemoglobin determination. Forensic Sci. Int. 37:
99-104 (1988).
6. K. Sato, K.Tamaki, H.Tsutsumi, H.Okajima, and Y. Katsumata.
Storage of blood for methemoglobin determination: comparison of
storage with a cryoprotectant at-30~ and without any additions
at -80~ or -196~ Forensic Sci. Int. 45:129-134 (1990).
7. I. Uchida, C. Tashido, Y.H. Koo, T. Mashimo, and I. Yoshiya. Carboxyhemoglobin and methemoglobin levels in banked blood.
J. Clin. Anesth. 2:86-90 (1990).
8. G.L. Moore, A. Zegna, M.E. Ledford, J.P. Huling, and R.M.
Fishman. Evaluation of methemoglobin formation during the
storage of various hemoglobin solutions. Artif. Organs 16(5):
513-518 (1992).
9. J.J. Mahoney, H.J. Vreman, D.K. Stevenson, and A.L. Van Kessel.
Measurement of carboxyhemoglobin and total hemoglobin by
five specialized spectrophotometers (CO-oximeters) in comparison
with reference methods. Clin. Chem. 39(8): 1693-1700 (1993).
10. A. Zwart, E.J.Van Kampen, and W.G. Zijlstra. Results of routines
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