PECCS, which affords direct acquisition of analytical data and storage of spectra, was used. Glass cuvettes of 1-cm light path and distilled water as sample blank ...
Journal of Analytical Toxicology, Vol. 26, March 2002
Determination of Methemoglobin and Total Hemoglobin in ToxicologicalStudiesby Derivative Spectrophotometry A. Cruz-Landeira, M.J. Bal, O. Quintela, and M. t6pez-Rivadulla Forensic Toxicology Service, Legal Medicine Institute, University of Santiago of Compostela, San Francisco s/n, 15705.Santiago of Compostela, Spain
Abstract ] A method for the determination of methemoglobin in the presence of other hemoglobin subforms (i.e., oxy-, deoxy-, and
carboxyhemoglobin) by use of derivative spectrophotometry is proposed. The method, which uses the first-derivative of the spectrum at 645 nm, is straightforward and expeditious, so it is of special interest to forensic toxicology laboratories as it allows the simultaneous determination of the methemoglobin saturation percentage and the hemoglobin concentration. This facilitates interpretation of the results and provides a better understanding of the significance of methemoglobin saturation in specific cases. Based on an analysis of interferences, the presence of other hemoglobin subforms or of endogenous components of plasma does not detract in any way from the performance of the method.
quently found in blood (i.e., oxyhemoglobin,deoxyhemoglobin, and carboxyhemoglobin).The method uses the first derivative of the absorption spectrum for the sample. Potential interferences from other frequently reported components including bilirubin, lipids, and Methylene Blue (a therapeutic agent for methemoglobin) were investigated. The method allows one to determine the methemoglobin saturation percentage and the total amount of hemoglobin, which facilitates interpretation of the analytical results.
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-
A PerkinElmer Lambda 2 ultraviolet-visible (UV-VIS)spectrophotometer interfaced to a computer running the software PECCS, which affords direct acquisition of analytical data and storage of spectra, was used. Glass cuvettes of 1-cm light path and distilled water as sample blank were employed. An Izasa STKS autoanalyzer was used to determine hemoglobin.
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.
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67
Journal of AnalyticalToxicology,Vol. 26, March 2002
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-
Table I. Compositionof SolutionsContaining Increasing Amounts (%) of MetHb MetHb % 0 2 4 6 8 10 20 40 60 80 100
[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
Table II. Compositionof SolutionsContaining Variable Concentrations of Hemoglobin (MetHb)
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).
Results and Discussion Distilled water was used as the blood diluting medium because it was found to providestable solutions while resulting in osmotic lysis of red cells. In addition, aqueous solutions are easier to handle than those used by other authors (e.g., phosphate buffer, Sterox-E). A 1% dilution was found to be the most effectivewith a viewto examiningthe spectrophotometric absorption band on which the Studywas based. Higher dilutions resulted in decreased absorbance, and lower ones distorted the band of interest. Other authors (12,13) use dilutions as high as 1:1000; this entails operating at lower wavelengths (405-425 nm), where the hemoglobin molecule exhibits a much higher absorbance. Figures 1 and 2 show the absorbance spectra for OxyHb, COHb, DeoxyHb,and MetHb, and their first derivativesoverthe range 500-700 nm, respectively. Notwithstanding the strong overlap of bands in the absorbance spectra (Figure 1), only MetHb absorbs at 645 nm (D1),with a positive peak that is not affected by the other components (Figure 2). Some authors (12) use the band at approximately 400 nm (Soret region) to quantitate MetHb because its absorbance is 50 times higher than in the 630 nm region. We discardedthis region becauseof the strong overlap between the spectra for the different hemoglobin subforms. In addition, some of the substances studied (e.g., bilirubin, which exhibits an absorbance peak at 450 nm) might also have interfered in this region. As can be seen from the spectra of Figure 3 and the data of Table III, 1D64snm (the quantity used to determine methe-
Journal of Analytical Toxicology, Vol. 26, March 2002
moglobin) was proportional to the MetHb content at a given concentration of hemoglobin. The corresponding linear regression equation was Y-- 0.025X + 0.060 (r = 0.9998) where Yis the ]D64snmvalue andX the MetHb percent content. Table III. Quantitation of MetHb in Solutions Containing Increasing Concentrations of this Hemoglobin Subform MetHb % 0 2
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
The precision of the method was studied at three different MetHb levels (i.e., 20, 55, and 100%). The coefficient of variation never exceeded 2.92 (n = 25). The limit of detection could not be determined as all samples contained some methemoglobin. Instead, the basal MetHb percentage was determined in 79 samples from blood donors and found to be 0.5% (SD = 0.0234). At a given MetHb saturation percentage, peak D 1 was proportional to the hemoglobin concentration. This is clearly apparent from Figure 4 and Table IV, which show the 1D645nm values for solutions of increasing concentrations of Hb 100% saturated with MetHb (the solutions in Table II). The corresponding linear regression equation for the hemoglobin concentration (g/dL) versus ]D64snmplot is Y2= 0.1999;(2 + 0.08 (r = 0.9999) where )'2 is the ]D64snmvalue and)(2 the real hemoglobin concentration (g/dL) in the sample concerned. The results were compared with those provided by a Coulter STKS autoanalyzer for 15 fresh blood samples from donors. Table V lists the hemoglobin concentrations provided by the proposed method (rightmost column) and those obtained with
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
!
u
~,O
O,MM Qi.I
i
m.O
~,0
i
M,O
nm
rim
Figure 1, Absorbancespectrafor OxyHb (A), COHb (g), DeoxyHb (C), and MetHb (D).
Figure 3. D1 spectra for solutions containing a constant concentration of Hb (1.3 mg/mL) and increasing amounts of MetHb (0, 20, 40, 60, 80, and 100%). See the increasing value for the 645 nm positive peak.
'" 5 . i
"1
J
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
Figure 2. Dn spectra for OxyHb (A),COHb (B), DeoxyHb(C), and MetHb (D),
Figure 4. D 1 spectra for solutions containing increasing concentrations of hemoglobin as MetHb (100% saturation).
69
Journal of Analytical Toxicology, Vol. 26, March 2002
the method used by the Clinical Analysis Laboratory (central column). As can be seen from Figure 5, the results of both methods are linearly correlated. The proposed method allows one not only to determine the proportion of methemoglobin but also to calculate the real concentration of hemoglobin in each sample and hence predict the functional implications of such a proportion in specific cases. This can be used to determine total hemoglobin in toxicological laboratories, where an autoanalyzer is rarely available. In addition, the proposed method surpasses the standard methods based on the addition of KCN typically used by clinical laboratories; in fact, it avoids the use of this reagent and hence its toxic effects. In practice, processing an unknown sample by using the proposed method involves the step sequence depicted in Scheme I. The proportion of MetHb, X, can be calculated from a simple rule of three:
The hemoglobin concentration in the working solution can be calculated by substituting the value of B into Eq. 2 (Y2 = 0.1999)(2 + 0.008): B = 0.19992(2 + 0.008 )(2 (g/dL) = (B - 0.008) / 0.1999
For example, if the "A value" for an unknown is 0.7890, and after the saturation of the sample with potassium ferricyanide the "B value" (the 100% of metHb for the sample) is 2.650, then: %MetHb = (0.7890 x 100)/2.650 = 78.90/2.650 = 29.7% of MetHb in the sample. [Hb] g/dL = (2.650 - 0.008)/0.1999 = 2.642/0.1999 --- 13.2 g/dL of Total Hb in the sample.
~' 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
Table V. Comparisonof the ResultsProvidedby the ProposedD1-Methodand the Coulter Method Sample
[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
% MetHb (X) = 100 xA/B
y = 0.9634X r = 0.9841
16
.:3:. 8 ~10
=/
0/0
6 6.0
I
I
I
I
I
i
8.0
10.0
12.0
14.0
16.0
18.0
[Hb] Coulter (g/dL) Figure5. Correlation of the results obtained in the determination of the hemoglobin concentration usingthe Coulter STKSmethod and the proposed (DI) method.
, •
Dilute 0.1 mL of the unknown blood sample until 10 mL with distilled water
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
SchemeL Procedure used to calculate the MetHb and Hb contents in a real sample.
Journal of Analytical Toxicology,Vol. 26, March 2002
Interferences
Other hemoglobin subforms. Any spectral interference from carboxyhemoglobin,oxyhemoglobin,or deoxyhemoglobinwas discarded earlier in establishing the most suitable operating conditions. As can be seen from Figure 2, which shows the D1spectra for these hemoglobin subforms and that for methemoglobin, the spectra for COI-Ib,OxyHb,and DeoxyHbdo not interfere with the determination of MetHb based on the first derivative of the spectra in the region of interest (645 nm). This was why parameter 1DB45nmwas chosen to quantitate MetHb. Potassium ferricyanide. Becausepotassium ferricyanidegives colored aqueous solutions, it is essential to obtain their spectra in order to discard potential interferences with the measurement of MetHb as it is used to saturate samples in the proposed method. A potassium ferricyanide solution at the usual concentration (10 mg/mL) did not interfere with the determination of MetHb as its Dl-spectrum was fiat in the measuring region (645 nm). Bilirubin. In order to determine the potential interference of bilirubin with the determination of MetHb from D], a solution containing a bilirubin concentration equivalentto that in blood from a patient with severe jaundice (22 mg/dL) was prepared and its spectrum recorded. Although the D1 spectrum exhibits a relativelystrong peak in the band at 500 nm (specifically,at approximately480 nm), its is virtually fiat between 500 and 700 nm. We quantitated the potential interference by adding increasing concentrations of bilirubin from 0 to 50 mg/dL to a blood samplewith a basal MetHb concentration of 0.5%. The interference was found to be quite weak at bilirubin concentrations up to 40 mg/dL, which increased the MetHb saturation percentage to 0.8%. Abovesuch a concentration, the interference was substantial (e.g., it raised the MetHb saturation percentage to 1.2% at 50 mg/dL). According to Zwart et al. (10), the strongest interferences are observed in blood with a high proportion of Hb F, which often contains a high enough bilirubin concentration to simulate an appreciable proportion of MetHb. The difficultyof obtaining specimensof this type prevented us from confirming this assertion except in fetal blood, which exhibited no signs of interference. Methylene Blue. It was important to establish the potential influence of Methylene Blue on the determination of the hemoglobin subforms studied as this dye is the treatment of choice for methemoglobinemia,so the need frequentlyexists to measure one substance in the presence of the other. To some authors (13), the presence of MethyleneBlue can result in significant errors in the conventional spectrophotometric determination of MetHb. The D1 spectrum for an aqueous solution of MethyleneBlue at over the wavelength range 500-700 nm was foundto exhibit a maximum at 679 nm and a minimum at 645 nm that bore a more or less constant relationship to each other. This suggests that Methylene Blue interferes severely with the determination of methemoglobin. However, taking into account that therapeutic dose of the dye is 1-2 mg/kg, its concentration will normally lie below28 mg/mL.No significant interference was observed over the concentration range tested (0-50 mg/mL) as the increase in the MetHb saturation percentage never exceeded 0.2%.
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.
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Journal of Analytical Toxicology,Vol. 26, March 2002 determination of clinically significant hemoglobin derivatives by multicomponent analysis. Clin. Chem. 32(6): 972-978 (1986). 11. M.L. Shih, W.D. Korte, and C.R. Clark. Multicomponent spectroscopic assay for hemoglobin and ferrihemoglobin species in methemoglobin treatment of cyanide poisoning. J. Anal. Toxicol. 21:543-547 (1997). 12. A. Taulier, R Levillain, and A. Lemonnier. Determining methemoglobin in blood by zero-crossing-point first derivative spec-
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trophotometry. Clin. Chem. 33(10): 1767-I 770 (1987). 13. J. Dotsch, S. Demirakca, D. Hahn, N. Katz, P.G. Kuhl, and W. Rascher. Accuracy of methemoglobin measurements: comparison of six different commercial devices and one manual method. Crit. Care Med. 27(6): 1191-I 194 (I 999). Manuscript received August 14, 2000; revision received January 22, 2001.
