The Spectrophotometric Measurement of Carboxyhemoglobin

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

ABSTRACT A 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.

Introduction etry.1’7’9’12’13 Of the various techniques men Carbon monoxide is the most frequently tioned, the procedure involving visible spec encountered gaseous poison. It binds readily trophotometry is considered to be highly with hemoglobin and thus interferes with sensitive and the most practical for use in oxygen transport. A rapid and accurate lab the routine clinical laboratory. Thus, such a oratory procedure for the detection of carb method is described in detail. It is realized oxyhemoglobin can be of great help to the that gas chromatographic techniques have physician in the successful diagnosis and gained in popularity; however, the unavail treatment of carbon monoxide poisoning. ability of the necessary instrumentation in Several approaches have been used for most clinical laboratories limits the use of the detection and quantitation of carbon this technique. monoxide in biological fluids. 2 The most Spectrophotometric methods are based popular procedures involve ( 1 ) gasometry, on absorbance measurements at 2 or 3 spe after the release of CO from the hemoglobin cific wavelengths before or after treatment complex, (2) gas chromotography4 ’1 1 (3) with reducing agents (figures 1 A and IB ). micro diffusion, involving the reduction of The methods published have the drawback, palladium chloride, (4) infrared spectro however, that other hemoglobin derivatives photometry3 and (5) visible spectrophotom or bilirubin cause interference. The method

3 6 SPECTROPHOTOMETRIC MEASUREMENT OF CARBOXYHEMOGLOBIN 3 7 presented8 is free of bilirubin interference Procedure and the standard curve is linear over the 1. One hundred /¿I of whole heparinized entire analytical range. blood are added to 25 ml of 0.4 percent NH4 OH. The solution is mixed and allowed Principle to stand for 2 minutes. A dilute hemolysate of blood is treated 2. Three ml of NH4OH and 3 ml of the with sodium dithionite which reduces met- hemolysate respectively are transferred into hemoglobin and oxyhemoglobin but does 1 cm cuvets. (The sample is analyzed in not affect carboxyhemoglobin. The absorb duplicate). ance of this solution is measured at 541 and 3. Ten mg of sodium dithionite are added 555 nm, the absorbance ratio of A541 nm/ to the cuvets. The cuvets are covered with A555 nm is calculated and the percent car Parafilm and gently inverted 10 times. boxyhemoglobin is determined from the 4. Exactly 5 minutes after the addition calibration chart. of dithionite to the sample, the absorbance at 541 and 555 nm is read against the R eagents NH 4O H blank. (If a number of samples NH±OH (0.4 percent). Approximately 16 are analyzed, the addition of the reducing ml of concentrated NH4OH are diluted to agent is spaced so that each can be read one 1 with deionized water. This solution after exactly 5 minutes). is stable. 5. The ratio of the absorbance at 541/ 555 nm is calculated and the percent car Sodium Hydrosulfite (sodium dithionite), boxyhemoglobin is determined from the AR. Ten mg portions of sodium dithionite calibration chart. are preweighed, placed into individual Note: For confirmation and for the pur small test tubes and the test tubes stoppered pose of record, the sample without and or covered with Parafilm. with dithionite may be scanned between Carbon monoxide. Commercial source.* 450 and 600 nm. (Between scans, the re Oxygen, C. P. corder paper is returned to the starting position of the first scan. The second scan, Special Apparatus after-dithionite, is recorded over the first A narrow band pass ( < 2 nm) spectro scan). Examples of scans for samples con photometer with 1 cm cuvets is required, taining 0 and 1 0 0 percent carboxyhemo although the use of a recording spectro globin, before and after the addition of photometer with the same specifications is dithionite, are shown in figures 1C and ID, desirable. The procedure listed was per respectively. Figures 2A and 2B show formed on a Beckman DB Recording Spec spectra obtained from samples with normal trophotometer. and increased carboxyhemoglobin content. It is imperative that the spectrophotom Preparation of the Standard Curve eter used is checked regularly for wave Caution—A fume hood should be used length and spectrophotometric accuracy10 with appropriate calibrating filters (e.g. when working with carbon monoxide gas. 1. Twenty ml of heparinized blood are NBS Reference Material 930) and with collected from a healthy non-smoker. liquid photometric standards (e.g. NBS 2. Four ml portions of the fresh, hepa Reference Material 931). rinized blood sample are transferred into * Lecture bottle, Matheson Gas Products, Div. each of two 125 ml separatory funnels. of Will Ross, Inc. These samples are treated with pure oxy 38 TIETZ AND FIEBECK

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 car boxyhemoglobin and 100 percent oxygenated hemo globin after treatment with sodium dithionite. Note that there is no change in the spectrum of carboxyhemoglobin while the change of oxygenated hemoglobin into deoxy genated hemoglobin re 600 560 520 480 nm600 560 520 480 nm sults in a significant m m change in the absorption spectrum. C. Spectral curves of 100 percent oxygenated hemoglobin before and after treat ment with sodium dithio nite. D. Spectral curves of 100 percent carboxy hemoglobin before and after treatment with so dium dithionite. Note that the spectral curves before and after dithionite treat ment are identical. (In strument: Beckman DB Recording Spectropho tometer ) 600 560 520 480 nm 600 560 520 480 nm gen and pure carbon monoxide respect then analyzed immediately in triplicate ac ively for 15 minutes while the funnels are cording to the procedure given and used gently rotated. After the addition of the for the establishment of the 0 and 100 per respective gas, the separatory funnels are cent carboxyhemoglobin calibration points. closed and rotated gently for an additional The ratios of the absorbance at 541/555 15 minutes. The fully saturated samples are nm for the 0 and 100 percent carboxyhemo-

F i c u r e 2. A. Spectrum of patient’s sample con taining 2 percent car boxyhemoglobin. B. Spec trum of patient’s sample containing 44 percent car boxyhemoglobin .

600 560 520 480nm 600 560 520 480nm SPECTROPHOTOMETRIC MEASUREMENT OF CARBOXYHEMOGLOBIN 3 9

Note: A sample calibration curve, ob tained 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 Notes and Discussion A fresh heparinized blood sample is the most desirable specimen. If the sample can not 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 pe riods of time may contain interfering pig F ig u r e 3. Example of a standard curve for the ments and therefore is unsuitable for this conversion of the 541/555 nm absorbance ratio to percent carboxyhemoglobin saturation (present analysis. method ). Methemoglobin is reduced by the dithio nite and therefore is not a source of inter globin samples are plotted and a line is ference. drawn between the two points (figure 3). The procedure recommends a 250-fold The ratios obtained in our laboratory for dilution of the sample so that the absorb the 0 and 1 0 0 percent carboxyhemoglobin ance reading at 541 nm can be taken in the were 0.825 ± 0.005 and 1.225 ± 0.005, re absorbance range of 0.2 to 0.5. If the hemo spectively. globin content of the sample is extremely 3. For the preparation of intermediate high or low, the dilution should be ad standards, the funnel containing the 1 0 0 justed so that the absorbance reading at percent carboxyhemoglobin is filled with 541 nm falls in this range. nitrogen gas and rotated for 5 minutes. The reduction of hemoglobin by sodium Treatment with nitrogen removes the phys dithionite has been studied extensively by ically dissolved CO from the sample but a Dalziel and O’Brien. 6 The reaction is very small amount of carbon monoxide will also dissociate from hemoglobin. The exact car boxyhemoglobin 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 nitro gen 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 ob tained. These points should fall on the line drawn for the fully saturated samples, since F ig u r e 4. Example of a standard curve based the curve is linear over the entire range on the method of Klendshoj et al.8 Note the non- (figure 3). linearity of this curve. 4 0 TIETZ AND FIERECK complex and numerous side reactions can et al, 9 in our hands, gave a coefficient of occur. Under the conditions of the de variation of 4.3 and 4.9 percent respec scribed procedure, the absorbance ratio re tively. mains nearly constant from 4 to 8 minutes after addition of sodium dithionite; how Discussion of Alternate ever, to insure uniformity, the readings are Spectrophotometric taken after exactly 5 minutes. M ethods Spectrophotometric and wavelength ac The absorption curve of oxygenated curacy must be maintained at all times hemoglobin has maxima at 577 and 541 nm. since one of the absorbance readings is As oxygenated hemoglobin is transformed taken at the down slope of the absorption into carboxyhemoglobin, the two absorption curve. A 5 nm downward shift or a 5 nm peaks shift towards 571 and 536 nm, the upward shift in the wavelength may cause peaks characteristic of carboxyhemoglobin.. an error in the A541/A555 nm ratio of 6 Inspection of the spectral scans of a diluted and 1.2 percent, respectively. In the pro hemolysate of blood allows qualitative de cedure by van Kemper and Klouwer, 1 2 a tection of large amounts of carboxyhemo similar 5 nm downward or upward shift globin, but detection of moderate to small in the wavelength can cause an error in amounts is difficult with this qualitative the A540/A579 nm ratio of 35 and 19 per method. cent, respectively. Because of the high de The quantitative spectrophotometric pendency of the latter method on wave method of Amenta1 is based on the deter length accuracy, it seems unsuitable for mination of the absorbance ratio, A575- routine laboratory use. A560 nm/A498 nm, of a hemolysate. The It is important that the procedure for the interference by other hemoglobin deriva preparation of the standard curve be fol tives as well as the interference by bilirubin lowed precisely. Samples saturated with (table I) limits the usefulness of this pure carbon monoxide are suitable for method. establishing the 1 0 0 percent carboxyhemo- Klendshoj et al9 suggest the determination globin saturation point. These samples can of the absorbance ratio of A555/A480 nm not be used for the determination of inter after treatment of the hemolysate with so mediate points of the calibration curve dium dithionite. This method has the dis since dissolved carbon monoxide (not hemo advantages that the standard curve is non globin bound) and small CO gas bubbles linear (figure 4), and that bilirubin, when trapped in the viscous sample, give higher present in a high concentration, gives a CO-concentrations than expected on the positive interference (table I). Bilirubin basis of a simple dilution. Treatment of interference can be reduced by washing samples with nitrogen, following saturation the cells with saline prior to analysis; how with carbon monoxide, will flush out the ever, some dissociation of the carbon excess dissolved CO but, at the same time, monoxide from hemoglobin causes a lower some CO will dissociate from the hemo ing of the results. globin complex, thus reducing the degree In a similar method, van Kampen and of saturation. Klouwen1 2 recommend the determination The coefficient of variation for a blood of carboxyhemoglobin by the absorbance sample containing approximately 40 percent ratio of nm after reduction of other carboxyhemoglobin, analyzed 15 times, by A579 the present method was 2.4 percent where interfering hemoglobin derivatives with as the methods of Amenta1 and Klendshoj dithionite. The method has the disadvan SPECTROPHOTOMETRIC MEASUREMENT OF CARBOXYHEMOGLOBIN 41

TABLE I

Added Amenta1 Klendshof Present Method Bilirubin A575-A560 A541 , , . A555. After ^ reduction , • , aftei• reduction in mg/100 ml A498 A. 4o(J 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 Resumé of Clinical Interpretations2 5 540 nm, and especially the point at 579 nm, Toxic symptoms, such as shortness of is located on a very steep portion of the breath, begin to appear when the carboxy curve which could result in gross errors if hemoglobin concentrations are above 1 0 wavelength accuracy is not very carefully percent; values of 25 to 30 percent cause controlled (See Procedure Notes and Dis commencement of the major symptoms of cussion). carbon monoxide poisoning such as severe headache, irritation, fatigue and disturb Sources o f Error ance of judgment. Levels of 60 to 70 per The major source of error in this11 and cent cause unconsciousness, respiratory other similar methods9 ’ 12 is that sulfhemo- failure and death if exposure is prolonged. globin interferes. Sulfhemoglobin is not Levels of 80 percent or above are rapidly affected by reducing agents and although fatal. two of its absorption peaks are near those Toxicity is influenced by extraneous fac of carboxyhemoglobin, sulfhemoglobin has tors such as the general health of the in an additional peak at 620 nm which can be dividual, hemoglobin content, time of ex used for its quantitation. 7 In cases of carbon posure and degree of activity. monoxide poisoning, it is generally assumed Owing to the high affinity of carbon that sulfhemoglobin is not present and that monoxide for hemoglobin, carboxyhemo only the two pigments, carboxyhemoglobin globin levels can accumulate as a result of and oxyhemoglobin, are present. In reality, repeated or prolonged exposure to carbon other interfering pigments may be present monoxide. and could reduce the accuracy of this At high oxygen tensions, carbon monoxide method. is readily replaced from hemoglobin by oxy gen. Thus, patients treated with oxygen Normal Range prior to the collection of the blood sample Ideally, the blood should contain no may show a normal carboxyhemoglobin carbon monoxide, however, levels of 0.5 to level even after severe carbon monoxide 2 percent carboxyhemoglobin are generally poisoning. found in non-smokers from rural areas and References values up to 5 percent in non-smokers from an urban environment. Heavy smokers may 1. Am enta, J.: The spectrophotometric deter mination of carbon monoxide in blood. Stan have carboxyhemoglobin values that range dard Methods of Clinical Chemistry 4:31-38, up to 9 percent. 1963. 4 2 TIETZ AND FIERECK

2. B l a n k e , R. V.: Carbon monoxide. Funda using the A541 nm/A555 nm ratio after the mentals of Clinical Chemistry, Tietz, N. W., addition of sodium dithionite. Unpublished in ed. W. B. Saunders, Philadelphia, pp. 835- formation. 884, 1970. 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 , 3. Coburn, R. F.: Infrared method for the mea A.: The spectrophotometric determination of surement of blood carboxyhemoglobin. Hemo carbon monoxide. J. Biol. Chem. 183:297-303, globin. Its Precursors and Metabolites, Sunder- 1950. man, F. W. and Sunderman, F. W., Jr., eds. 10. R a n d , R . N.: The standardization of spectro J. B. Lippincott, Philadelphia, pp. 67-69, 1964. photometers. Proceedings of the International 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 , Seminar and Workshop on Enzymology spon J. D.: Determination of carbon monoxide in sored by University of Health Sciences/Chicago blood by gas chromatography. Clin. Chem. 14: Medical School and Mount Sinai Hospital 162-171, 1968. Medical Center, Chicago, May 1972, pp. 3- 5. C b r r y , A. S.: Poison Detection in Human 2 5 -3 -3 1 . Organs, Charles C Thomas, Springfield, pp. 11. Su n d s t r ö m , G.: Blood carboxyhemoglobin. 28-32, 1963. Results with conventional standards compared 6. D a l z i e l , K. a n d O ’B r i e n , J. R. P.: Side reac with those with a submicroliter reference of tions in the deoxygenation of dilute oxyhemo gaseous CO. Clin. Chem. 18:188-192, 1972. globin solutions by sodium dithionite. Biochem. 12. v a n K ä m p e n , E. J. a n d K l o u w e n , H.: Spec J. 67:119-124, 1957. trophotometric determination of carboxyhemo 7. D u b o w s k i, K. M.: Measurements of hemo globin. Rec. Trav. Chim. Paysbas. 73:119-128, globin derivatives. Hemoglobin: Its Precursors 1954. and Metabolites. Sunderman, F. W. and Sun 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.: derman, F. W., Jr., eds. J. B. Lippincott, Determination of hemoglobin and its deriva Philadelphia, pp. 49-60, 1964. tives. 6. Spectrophotometric determination of 8. F i e r e c k , E. A. a n d T i e t z , N. W.: Modified carboxyhemoglobin. Advances in Clinical Klendshoj et al. method for the spectrophoto- Chemistry 8:170-176, 1965. Sobotka, H. and metric determination of carboxyhemoglobin Stewart, C. P., eds., Academic Press, NY.