Determination of Carbon Monoxide in Blood Jack L . Lambert, Reginald R. Tschorn, and Philip A. Hamlin’ Department of Chemistry, Kansas State University, Manhattan, Kans. 66502
DETERMINATION OF CARBON MONOXIDE in blood (carboxyhemoglobin) is important in the diagnosis of carbon monoxide poisoning. A survey of several manuals on clinical laboratory methods (1-4) showed that variations of four basic quantitative methods are most frequently recommended : the differential spectrophotometric method for oxyhemoglobin and carboxyhemoglobin of Hartridge (5); the spectrophotometric determination of carboxyhemoglobin following selective conversion of oxyhemoglobin to hemoglobin by hydrosulfite, Na2S204,as described by Klendshoj, Feldstein, and Sprague ( 6 ); the colorimetric or titrimetric method involving reduction of palladium(I1) chloride by carbon monoxide reported by Feldstein and Klendshoj (7); and the spectrophotometric method of Whitehead and Worthington (8) which involves precipitation of oxyhemoglobin in the presence of carboxyhemoglobin by heating a t p H 5.28. According to Jacobs (9), the spectrophotometric methods of Hartridge (5) and Klendshoj, Feldstein, and Sprague ( 6 ) are accurate only when high concentrations of carbon monoxide are present. H e considered the manometric method of Van Slyke and Salvesen (10) and the volumetric method of Scholander and Roughton (11) to be accurate but demanding considerable skill and practice. H e recommended the spectrophotometric method of Lawther and Apthorp (12) and the pyrotannic acid method of Sayers et al. ( I S , 14). Sunshine (15) recommended the microdiffusion method of Feldstein and Klendshoj (7). Wilson and Jay (16) favored a gas chro-
3 mm
Present address, Department of Chemistry, Panhandle State College, Goodwell, Okla. (1) “Bray’s Clinical Laboratory Methods,” 7th ed., revised by J. D. Bauer, P. G. Ackerman, and G. Toro, C. V. Mosby Co., St. Louis, Mo., 1968, pp 101-5, 691-2. (2) “Gradwohl’s Clinical Laboratory Methods and Diagnosis,” Vol. 1, 7th ed., S. Frankel, S . Reitman, and A. C. Sonnenwirth, Ed., C. V. Mosby Co., S t . Louis, Mo., 1970, pp 303-5,397-9. (3) M. J. Lynch, S. S. Raphael, L. D. Mellor, P. D. Spare, and M. J. H. Inwood, “Medical Laboratory Technology and Clinical Pathology,” 2nd ed., W. B. Saunders Co., Philadelphia, Pa., 1969, pp 343-6. (4) “Fundamentals of Clinical Chemistry,” N. W. Tietz, Ed., W. B. Saunders, Co., Philadelphia, Pa., 1970, pp 835-40. (5) H. Hartridge, J . Plzysiol. (London),57,47 (1922). (6) N. C. Klendshoj, M. Feldstein, and A. L. Sprague, J . Biol. Chem., 183,297 (1950). (7) M. Feldstein and N. C. Klendshoj,J. Forensic Sci., 2,39 (1957). (8) T. P. Whitehead and S. Worthington, Cliii. Cliem. Acta, 6, 356 (1961). (9) M. B. Jacobs, “The Analytical Toxicology of Industrial Inorganic Poisons,” Interscience Publishers, New York, N. Y., 1967, pp 713-17,840-2. (IO) D. D. Van Slyke and H. A. Salvesen, J . Biol. Cliem., 40, 103 ( 1919). (11) P. F. Scholander and F. J. W. Roughton, ibid., 148,551 (1943). (12) P. J . Lawther and G. H. Apthorp, Brit. J . h d . Med., 12, 326 I1 955). ( 1 3 ) k.’R.Sayers and W. P. Yant, U.S. Pub. Health Sercice Repr., 790 (1922). (14) R. R. Sayers, W. P. Yant, and G. W. Jones, ibid., 872 (1924). (1 5) I. Sunshine, “Handbook of Analytical Toxicology,” Chemical Rubber Co., Cleveland, Ohio, 1969, pp 1015-18. (16) R. H. Wilson and B. E. Jay, Clii7. Res., 8,92 (1960).
Figure 1. Modified Conway microdiffusion cell with plunger matographic method for carbon monoxide in complex gas mixtures. We have adapted the colorimetric method of Lambert and Hamlin (17) to the analysis of carboxyhemoglobin, using chicken blood in a modified Conway microdiffusion cell. EXPERIMEiWAL
Microdiffusion Cell. The cell shown in Figure 1 was constructed to permit lowering of the reagent solution into the light path of a Bausch and Lomb Spectronic 20 spectrophotometer. A Teflon (Du Pont) rod was machined with air-tight flanges to fit the upper and lower precision-bore tubes, and a slot was cut in the lower portion t o coincide with the light path of the spectrophotometer. Ports equipped with rubber septums were designed t o permit injection by syringe of the reagent into the inner portion of the cell, and injection of the blood sample and the 10% sulfuric acid solution into the outer portion of the cell. Reagent. A n aqueous solution of tetrachloropalladate(II), ethylenediaminetetraacetatoferrate(II1) and molybdate anions, (17) J. L. Lambert and P. A. Hamlin, A/7tr[.Lett., 4,745 (1971). ANALYTICAL CHEMISTRY, VOL. 44, NO. 8, JULY 1972
0
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A
0.60
Table I. Data for Calibration Curve Saturation,
0.50
W 0.40 V
z Q
m 0.30
(z
8m Q
0.20
Mean
Range
Standard deviation
0 25 50 75 100
0.022 0.156 0.308 0.454 0.598
0.015-0.028 0.145-0.168 0.298-0.315 0.438-0.470 0.585-0.610
0.005 0.009 0.007 0.013 0.009
D
0.1 0
E 0.00 0
7z
5
10
15
20
25
30
35
40
45
50
55
60
TIME ( I N MINUTES)
Figure 2. Rate of color development in reagent Per cent saturation of carbon monoxidein blood: Curve A = 100 %; Curve B = 75%; Curve C = 50%; Curve D = 25%; Curve E =
The reaction is essentially complete at all concentrations after 1 hour, although usable quantitative results would be possible with decreased sensitivity after 30 minutes. Table I shows the results obtained at five concentrations after 1-hour reaction time. The equation for the straight line calibration curve is
A = 1.8 X 10-2 -t 5.8 X 10-aP
0%
and 1,lO-phenanthroline at pH 7.9 f 0.1 was prepared as described by Lambert and Hamlin (17). The reagent was prepared daily from stock solutions of the component compounds. Procedure. Ten milliliters of heparinized chicken blood, 12.50 =k 0.01 grams of hemoglobin per 100 ml, was equilibrated with pure carbon monoxide in a 125-ml flask for 30 minutes in a mechanical shaker at 41 "C. Dilutions of 75, 50, and 25% saturation were made with CO-free blood. To the inner compartment of the cell, 1.00 ml of reagent was added and lowered into the beam of the spectrophotometer by depressing the Teflon tube. The spectrophotometer was set at zero absorbance at 522 nm. The reagent solution was then raised into the inner compartment of the microdiffusion cell by pulling up the Teflon rod. By means of syringes, 2.00 ml of blood sample and 2.00 ml of 10% sulfuric acid were injected into the outer compartment of the cell. Absorbance measurements were made at 5-minute intervals for a period of 1 hour. Light in the visible region is necessary for the reagent to react at a maximum rate. In darkness, the readings were approximately one-half those made in the presence of light, Ordinary laboratory lighting was sufficient to effect maximum reactivity of the reagent. RESULTS
Figure 2 shows the relationship of time of reaction in the microdiffusion cell to absorbance produced in the reagent.
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where A is absorbance and P is hemoglobin in the blood.
concentration of carboxy-
DISCUSSION
Most colorimetric methods for the determination of carbon monoxide are limited by the low solubility of carbon monoxide in aqueous solutions. In microdiffusion cells, the reaction time is further slowed by the time necessary for complete diffusion of released carbon monoxide through the air space above the sample and reagent solutions. The method described here probably could be speeded up if stirring of the reagent and/or sample solutions were possible. An advantage of our method is the production of a soluble colored compound rather than the colloidal metal dispersions formed as a surface skin in methods involving the reduction of palladium(I1) compounds. No colloidal palladium metal was formed in our reagent when 1,lO-phenanthroline was used, although a small amount was observed when 2,2'-dipyridyl was used (17). RECEIVED for review January 10, 1972. Accepted March 14, 1972. Work supported in part by National Institutes of Health Grant FR-07036 and National Science Foundation Grant GP-22734.
