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 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.