A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e , Vol. 3 , No. 1 Copyright © 1973, Institute for Clinical Science

The Spectrophotom etric M easurement

of Carboxyhem oglobin

NORBERT W. TIETZ, Ph.D. AND ERMALINDA A. FIERECK, M.S.

Division of Clinical Chemistry, Mt. Sinai Hospital Medical Center,

and the University of Health Sciences the Chicago Medical School,

Chicago, IL 60608

ABSTRACTA simple, reliable and rapid method for the spectrophotometric determina­

tion of carboxyhemoglobin in fresh blood is described. A blood hemolysate is prepared by diluting a blood sample with 0.4 percent NH4 OH. Addition of sodium dithionite deoxygenates hemoglobin (and reduces methemoglobin if present) without affecting carboxyhemoglobin. Comparison of the 541/555 nm absorbance ratio for the hemolysate with the ratio obtained for solutions with known concentrations of carboxyhemoglobin serves as a means to determine the percent of carboxyhemoglobin present in the sample. Bilirubin and methemoglobin do not interfere with the procedure, and the standard curve is linear over the entire analytical range.

In troductionCarbon monoxide is the most frequently

encountered gaseous poison. It binds readily with hemoglobin and thus interferes with oxygen transport. A rapid and accurate lab­oratory procedure for the detection of carb­oxyhemoglobin can be of great help to the physician in the successful diagnosis and treatment of carbon monoxide poisoning.

Several approaches have been used for the detection and quantitation of carbon monoxide in biological fluids. 2 The most popular procedures involve ( 1 ) gasometry, after the release of CO from the hemoglobin complex, (2 ) gas chromotography4 ’1 1 (3 ) micro diffusion, involving the reduction of palladium chloride, (4 ) infrared spectro­photometry3 and (5) visible spectrophotom­

etry. 1 ’ 7 ’ 9 ’ 1 2 ’ 13 Of the various techniques men­tioned, the procedure involving visible spec­trophotometry is considered to be highly sensitive and the most practical for use in the routine clinical laboratory. Thus, such a method is described in detail. It is realized that gas chromatographic techniques have gained in popularity; however, the unavail­ability of the necessary instrumentation in most clinical laboratories limits the use of this technique.

Spectrophotometric methods are based on absorbance measurements at 2 or 3 spe­cific wavelengths before or after treatment with reducing agents (figures 1 A and IB ). The methods published have the drawback, however, that other hemoglobin derivatives or bilirubin cause interference. The method

3 6

S P E C T R O P H O T O M E T R IC M E A SU R E M E N T O F CA R BO XYH EM O G LO BIN 3 7

presented8 is free of bilirubin interference and the standard curve is linear over the entire analytical range.

Princip le

A dilute hemolysate of blood is treated with sodium dithionite which reduces met- hemoglobin and oxyhemoglobin but does not affect carboxyhemoglobin. The absorb­ance of this solution is measured at 541 and 555 nm, the absorbance ratio of A541 nm/ A555 nm is calculated and the percent car­boxyhemoglobin is determined from the calibration chart.

R eagents

NH±OH (0.4 percent). Approximately 16 ml of concentrated NH4OH are diluted to one 1 with deionized water. This solution is stable.

Sodium Hydrosulfite (sodium dithionite), AR. Ten mg portions of sodium dithionite are preweighed, placed into individual small test tubes and the test tubes stoppered or covered with Parafilm.

Carbon monoxide. Commercial source.*Oxygen, C. P.

Special ApparatusA narrow band pass ( < 2 nm) spectro­

photometer with 1 cm cuvets is required, although the use of a recording spectro­photometer with the same specifications is desirable. The procedure listed was per­formed on a Beckman DB Recording Spec­trophotometer.

It is imperative that the spectrophotom­eter used is checked regularly for wave­length and spectrophotometric accuracy10

with appropriate calibrating filters (e.g. NBS Reference Material 930) and with liquid photometric standards (e.g. NBS Reference Material 931).

* Lecture bottle, Matheson Gas Products, Div. of Will Ross, Inc.

Procedure

1. One hundred /¿I of whole heparinized blood are added to 25 ml of 0.4 percent NH4 OH. The solution is mixed and allowed to stand for 2 minutes.

2. Three ml of NH4OH and 3 ml of the hemolysate respectively are transferred into1 cm cuvets. (The sample is analyzed in duplicate).

3. Ten mg of sodium dithionite are added to the cuvets. The cuvets are covered with Parafilm and gently inverted 10 times.

4. Exactly 5 minutes after the addition of dithionite to the sample, the absorbance at 541 and 555 nm is read against the N H 4O H blank. (If a number of samples are analyzed, the addition of the reducing agent is spaced so that each can be read after exactly 5 minutes).

5. The ratio of the absorbance at 541/ 555 nm is calculated and the percent car­boxyhemoglobin is determined from the calibration chart.

Note: For confirmation and for the pur­pose of record, the sample without and with dithionite may be scanned between 450 and 600 nm. (Between scans, the re­corder paper is returned to the starting position of the first scan. The second scan, after-dithionite, is recorded over the first scan). Examples of scans for samples con­taining 0 and 1 0 0 percent carboxyhemo­globin, before and after the addition of dithionite, are shown in figures 1C and ID, respectively. Figures 2A and 2B show spectra obtained from samples with normal and increased carboxyhemoglobin content.

Preparation o f th e Standard Curve

Caution—A fume hood should be used when working with carbon monoxide gas.

1. Twenty ml of heparinized blood are collected from a healthy non-smoker.

2. Four ml portions of the fresh, hepa­rinized blood sample are transferred into each of two 125 ml separatory funnels. These samples are treated with pure oxy­

38 T IE T Z AND F IE B E C K

600 560 520 480 nm600 560 520 480 nmmm

600 560 520 480 nm 600 560 520 480 nm

gen and pure carbon monoxide respect­iv ely for 15 minutes while the funnels are gently rotated. After the addition of the respective gas, the separatory funnels are closed and rotated gently for an additional 15 minutes. The fully saturated samples are

F i g u r e 1. A. Spectral curves of 100 percent carboxyhemoglobin and 100 percent oxygenated hemoglobin. B. Spectral curves of 100 percent car­boxyhemoglobin and 100 percent oxygenated hemo­globin after treatment with sodium dithionite. Note that there is no change in the spectrum of carboxyhemoglobin while the change of oxygenated hemoglobin into deoxy­genated hemoglobin re­sults in a significant change in the absorption spectrum. C. Spectral curves of 100 percent oxygenated hemoglobin before and after treat­ment with sodium dithio­nite. D. Spectral curves of 100 percent carboxy­hemoglobin before and after treatment with so­dium dithionite. Note that the spectral curves before and after dithionite treat­ment are identical. (In ­strument: Beckman DB R ecord ing Sp ectrop h o ­tometer )

then analyzed immediately in triplicate ac­cording to the procedure given and used for the establishment of the 0 and 100 per­cent carboxyhemoglobin calibration points.

The ratios of the absorbance at 541/555 nm for the 0 and 100 percent carboxyhemo-

F i c u r e 2. A. Spectrum of patient’s sample con­taining 2 percent car­boxyhemoglobin. B. Spec­trum of patient’s sample containing 44 percent car­boxyhemoglobin .

600 560 520 4 80nm 600 560 520 4 8 0 nm

SP E C T R O P H O T O M E T R IC M E A SU R E M E N T O F CA R BO XYH EM O G LO BIN 3 9

F ig u r e 3. Example of a standard curve for the conversion of the 541/555 nm absorbance ratio to percent carboxyhemoglobin saturation (present method ).

globin samples are plotted and a line is drawn between the two points (figure 3). The ratios obtained in our laboratory for the 0 and 1 0 0 percent carboxyhemoglobin were 0.825 ± 0.005 and 1.225 ± 0.005, re­spectively.

3. For the preparation of intermediate standards, the funnel containing the 1 0 0

percent carboxyhemoglobin is filled with nitrogen gas and rotated for 5 minutes. Treatment with nitrogen removes the phys­ically dissolved CO from the sample but a small amount of carbon monoxide will also dissociate from hemoglobin. The exact car­boxyhemoglobin content of this sample is determined by the method described using the standard curve prepared in step # 2 .

Intermediate standards are prepared by mixing appropriate proportions of the nitro­gen treated sample with the oxygen treated sample.

4. Each of the diluted blood samples from step # 3 is analyzed in triplicate.

5. The calculated concentrations are then plotted against the absorbance ratios ob­tained. These points should fall on the line drawn for the fully saturated samples, since the curve is linear over the entire range (figure 3).

Note: A sample calibration curve, ob­tained by the method of Klendshoj et al, 9

utilizing the absorbance ratio A555/A480 nm after the addition of sodium dithionite is shown for comparison in figure 4.

Procedure N otes and D iscussion

A fresh heparinized blood sample is the most desirable specimen. If the sample can­not be analyzed immediately, it should be kept tightly stoppered, refrigerated and protected from light. Post mortem blood or blood that has been stored for long pe­riods of time may contain interfering pig­ments and therefore is unsuitable for this analysis.

Methemoglobin is reduced by the dithio­nite and therefore is not a source of inter­ference.

The procedure recommends a 250-fold dilution of the sample so that the absorb­ance reading at 541 nm can be taken in the absorbance range of 0.2 to 0.5. If the hemo­globin content of the sample is extremely high or low, the dilution should be ad­justed so that the absorbance reading at 541 nm falls in this range.

The reduction of hemoglobin by sodium dithionite has been studied extensively by Dalziel and O’Brien. 6 The reaction is very

F ig u r e 4. Example of a standard curve based on the method of Klendshoj et al.8 Note the non- linearity of this curve.

4 0 T IE T Z AND F IE R E C K

complex and numerous side reactions can occur. Under the conditions of the de­scribed procedure, the absorbance ratio re­mains nearly constant from 4 to 8 minutes after addition of sodium dithionite; how­ever, to insure uniformity, the readings are taken after exactly 5 minutes.

Spectrophotometric and wavelength ac­curacy must be maintained at all times since one of the absorbance readings is taken at the down slope of the absorption curve. A 5 nm downward shift or a 5 nm upward shift in the wavelength may cause an error in the A541/A555 nm ratio of 6

and 1.2 percent, respectively. In the pro­cedure by van Kemper and Klouwer, 1 2 a similar 5 nm downward or upward shift in the wavelength can cause an error in the A540/A579 nm ratio of 35 and 19 per­cent, respectively. Because of the high de­pendency of the latter method on wave­length accuracy, it seems unsuitable for routine laboratory use.

It is important that the procedure for the preparation of the standard curve be fol­lowed precisely. Samples saturated with pure carbon monoxide are suitable for establishing the 1 0 0 percent carboxyhemo- globin saturation point. These samples can­not be used for the determination of inter­mediate points of the calibration curve since dissolved carbon monoxide (not hemo­globin bound) and small CO gas bubbles trapped in the viscous sample, give higher CO-concentrations than expected on the basis of a simple dilution. Treatment of samples with nitrogen, following saturation with carbon monoxide, will flush out the excess dissolved CO but, at the same time, some CO will dissociate from the hemo­globin complex, thus reducing the degree of saturation.

The coefficient of variation for a blood sample containing approximately 40 percent carboxyhemoglobin, analyzed 15 times, by the present method was 2.4 percent where­as the methods of Amenta1 and Klendshoj

et al, 9 in our hands, gave a coefficient of variation of 4.3 and 4.9 percent respec­tively.

D iscussion o f A lternateSpectrophotom etricM ethods

The absorption curve of oxygenated hemoglobin has maxima at 577 and 541 nm. As oxygenated hemoglobin is transformed into carboxyhemoglobin, the two absorption peaks shift towards 571 and 536 nm, the peaks characteristic of carboxyhemoglobin.. Inspection of the spectral scans of a diluted hemolysate of blood allows qualitative de­tection of large amounts of carboxyhemo­globin, but detection of moderate to small amounts is difficult with this qualitative method.

The quantitative spectrophotometric method of Amenta1 is based on the deter­mination of the absorbance ratio, A575- A560 nm/A498 nm, of a hemolysate. The interference by other hemoglobin deriva­tives as well as the interference by bilirubin (table I ) limits the usefulness of this method.

Klendshoj et al9 suggest the determination of the absorbance ratio of A555/A480 nm after treatment of the hemolysate with so­dium dithionite. This method has the dis­advantages that the standard curve is non­linear (figure 4 ), and that bilirubin, when present in a high concentration, gives a positive interference (table I ) . Bilirubin interference can be reduced by washing the cells with saline prior to analysis; how­ever, some dissociation of the carbon monoxide from hemoglobin causes a lower­ing of the results.

In a similar method, van Kampen and Klouwen1 2 recommend the determination of carboxyhemoglobin by the absorbance

ratio of nm after reduction of otherA579interfering hemoglobin derivatives with dithionite. The method has the disadvan­

S P E C T R O P H O T O M E T R IC M E A S U R E M E N T O F C A R B O X Y H E M O G L O B IN 41

TABLE I

Added Bilirubin

in mg/100 ml

Amenta1 A575-A560

A498

KlendshofA555 ^ , •. After reduction A. 4o(J

Present MethodA541 , , ., aftei• reduction A555

Ratio Percent Ratio Percent Ratio Percent

0 0.695 (42) 2.43 (41) 1.00 (44)5.0 0.665 (40) 2.23 (57) 1.00 (44)

10.0 0.620 (35) 2.11 (70) 1.00 (44)15.0 0.584 (31) 1.90 (>100) 1.00 (44)

Effect of increasing concentrations of bilirubin on absorbance ratios (and calculated percent CO-Satura- tion) obtained with three different methods for carboxyhemoglobin. These analyses were performed simul- aneously on the same sample, before and after the addition of bilirubin.

tage, however, that the absorption point at 540 nm, and especially the point at 579 nm, is located on a very steep portion of the curve which could result in gross errors if wavelength accuracy is not very carefully controlled (See Procedure Notes and Dis­cussion).

Sources o f Error

The major source of error in this11 and other similar methods9 ’ 12 is that sulfhemo- globin interferes. Sulfhemoglobin is not affected by reducing agents and although two of its absorption peaks are near those of carboxyhemoglobin, sulfhemoglobin has an additional peak at 620 nm which can be used for its quantitation. 7 In cases of carbon monoxide poisoning, it is generally assumed that sulfhemoglobin is not present and that only the two pigments, carboxyhemoglobin and oxyhemoglobin, are present. In reality, other interfering pigments may be present and could reduce the accuracy of this method.

N orm al R ange

Ideally, the blood should contain no carbon monoxide, however, levels of 0.5 to2 percent carboxyhemoglobin are generally found in non-smokers from rural areas and values up to 5 percent in non-smokers from an urban environment. Heavy smokers may have carboxyhemoglobin values that range up to 9 percent.

R esu m é o f C linical In terpretation s2 5Toxic symptoms, such as shortness of

breath, begin to appear when the carboxy­hemoglobin concentrations are above 1 0

percent; values of 25 to 30 percent cause commencement of the major symptoms of carbon monoxide poisoning such as severe headache, irritation, fatigue and disturb­ance of judgment. Levels of 60 to 70 per­cent cause unconsciousness, respiratory failure and death if exposure is prolonged. Levels of 80 percent or above are rapidly fatal.

Toxicity is influenced by extraneous fac­tors such as the general health of the in­dividual, hemoglobin content, time of ex­posure and degree of activity.

Owing to the high affinity of carbon monoxide for hemoglobin, carboxyhemo­globin levels can accumulate as a result of repeated or prolonged exposure to carbon monoxide.

At high oxygen tensions, carbon monoxide is readily replaced from hemoglobin by oxy­gen. Thus, patients treated with oxygen prior to the collection of the blood sample may show a normal carboxyhemoglobin level even after severe carbon monoxide poisoning.

References1 . A m e n t a , J.: The spectrophotometric deter­

mination of carbon monoxide in blood. Stan­dard Methods of Clinical Chemistry 4:31-38,1963.

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2. B l a n k e , R. V.: Carbon monoxide. Funda­mentals of Clinical Chemistry, Tietz, N. W., ed. W. B. Saunders, Philadelphia, pp. 835- 884, 1970.

3. Coburn, R. F.: Infrared method for the mea­surement of blood carboxyhemoglobin. Hemo­globin. Its Precursors and Metabolites, Sunder- man, F. W. and Sunderman, F. W., Jr., eds. J. B. Lippincott, Philadelphia, pp. 67-69, 1964.

4. C o l l i s o n , H. A., R o d k e y , F. L., a n d O ’N e a l , J. D.: Determination of carbon monoxide in blood by gas chromatography. Clin. Chem. 14: 162-171, 1968.

5. C b r r y , A. S.: Poison Detection in Human Organs, Charles C Thomas, Springfield, pp. 28-32, 1963.

6. D a l z i e l , K. a n d O ’B r i e n , J. R. P.: Side reac­tions in the deoxygenation of dilute oxyhemo­globin solutions by sodium dithionite. Biochem. J. 67:119-124, 1957.

7. D u b o w s k i , K. M.: Measurements of hemo­globin derivatives. Hemoglobin: Its Precursors and Metabolites. Sunderman, F. W. and Sun­derman, F. W., Jr., eds. J. B. Lippincott, Philadelphia, pp. 49-60, 1964.

8. F i e r e c k , E. A. a n d T i e t z , N. W.: Modified Klendshoj et al. method for the spectrophoto- metric determination of carboxyhemoglobin

using the A541 nm/A555 nm ratio after the addition of sodium dithionite. Unpublished in­formation.

9. K l e n d s h o j , N., F e l d s t e i n , M., a n d S p r a g u e , A.: The spectrophotometric determination of carbon monoxide. J. Biol. Chem. 183:297-303, 1950.

10. R a n d , R . N.: The standardization of spectro­photometers. Proceedings of the International Seminar and Workshop on Enzymology spon­sored by University of Health Sciences/Chicago Medical School and Mount Sinai Hospital Medical Center, Chicago, May 1972, pp. 3 - 2 5 -3 -3 1 .

11. Su n d s t r ö m , G.: Blood carboxyhemoglobin. Results with conventional standards compared with those with a submicroliter reference of gaseous CO. Clin. Chem. 18:188-192, 1972.

12. v a n K ä m p e n , E. J. a n d K l o u w e n , H.: Spec­trophotometric determination of carboxyhemo­globin. Rec. Trav. Chim. Paysbas. 73:119-128, 1954.

13. v a n K ä m p e n , E. J. a n d Z i j l s t r a , W. G.: Determination of hemoglobin and its deriva­tives. 6. Spectrophotometric determination of carboxyhemoglobin. Advances in Clinical Chemistry 8:170-176, 1965. Sobotka, H. and Stewart, C. P., eds., Academic Press, NY.