Absorption Spectra of Respiratory Pigments

The hemoglobins and other respiratory pigments—like all pigments—differentially absorb various wavelengths of light. The pattern of absorption by a pigment when expressed as a function of wavelength, is known as an absorption spectrum (plural spectra). The absorption spectrum of a specific respiratory pigment (e.g., human hemoglobin) changes with the oxygenation or deoxygenation of the pigment, as shown in the accompanying figure. These changes are qualitatively evident to our eyes: We know, for example, that oxygenated hemoglobin (bright red) differs in color from deoxygenated hemoglobin (purple-red). By using quantitative light-absorption measurements, the percentage of heme groups that are oxygenated in blood can be determined. This is the principle behind the finger probes—known as pulse oximeters—that are used to monitor arterial blood oxygenation in hospital patients. Box Extension 24.1 explains how a pulse oximeter measures the percentage of oxygenated heme groups in arterial blood and why it is called a “pulse” oximeter.

Absorption spectra for fully oxygenated and fully deoxygenated human hemoglobin To measure absorption, light of each wavelength is passed through a hemoglobin solution of defined concentration and optical path length (in the case shown here, the concentration was 1 mM, and the light path through the solution was 1 cm long). The fraction of the incoming photon energy that fails to pass through the solution is measured. From the data, one calculates the extinction coefficient, which is a measure of the absorption of the light by the hemoglobin: A high extinction coefficient signifies high absorption. (After Waterman 1978.)

If you look at the figure, you will note that when red light at a wavelength of about 650 nm is passed through hemoglobin, there is a large difference in light absorption, depending on whether the hemoglobin is oxygenated or deoxygenated. The figure is based on studies of fully oxygenated and fully deoxygenated hemoglobin. In the blood of a person or other mammal, however, the hemoglobin is partly oxygenated and partly deoxygenated. Speaking more exactly, some of the O₂-binding heme groups in the blood hemoglobin molecules are oxygenated, whereas others are deoxygenated. To determine the percentage of heme groups that are oxygenated in blood, one needs—in effect—to measure the concentration of oxygenated heme groups and the concentration of deoxygenated heme groups. That is, one must measure two properties! Then, the percentage oxygenated can be determined by expressing the concentration of oxygenated heme as a percentage of the concentration of all heme (i.e., the sum of the oxygenated and deoxygenated heme). If one passes 650-nm red light through blood that is partly oxygenated and partly deoxygenated, the absorption of the light will depend on the concentrations of oxygenated and deoxygenated heme. The absorption, that is, will contain information on the two unknowns of interest. However, the rules of algebra dictate that with one measurement—the absorption of red light—one cannot calculate two unknowns.

To calculate two unknowns, one must have two pertinent measurements. At infrared wavelengths near 950 nm, there is a second broad range of wavelengths where the absorption spectra of oxygenated and deoxygenated hemoglobins are different; at these infrared wavelengths, oxygenated heme absorbs to a greater extent than deoxygenated (opposite to the case at 650 nm). Electromagnetic radiation is invisible at infrared wavelengths. Nonetheless, if one passes 950-nm electromagnetic radiation through blood, the absorption of the radiation can be measured, and—as with the absorption of red light—it contains information on the concentrations of oxygenated and deoxygenated heme.

Oximeters simultaneously measure absorption by the blood of both red light at about 650 nm and infrared electromagnetic radiation at about 950 nm. In this way they obtain two measures of absorption. From such data, the two unknowns of interest—the concentrations of oxygenated and deoxygenated heme—can be calculated, and from these results the percentage of oxygenated heme groups can be calculated. The actual calculations are not carried out exactly as portrayed here. Nonetheless, the principles we’ve discussed explain the fundamental reason why measures must be taken at two different wavelengths.

In some disease states, a portion of the blood hemoglobin can exist in one or both of two pathological hemoglobin forms that are incapable of binding with O₂: methemoglobin (hemoglobin in which the iron is fully oxidized) and carboxyhemoglobin (hemoglobin bound to carbon monoxide). These forms of hemoglobin exhibit distinctive absorption spectra, different from the spectra of oxyhemoglobin and deoxyhemoglobin. To measure all four forms of hemoglobin, one would need to use four wavelengths of light or infrared radiation and obtain four separate measures of absorption by the blood. Basic oximeters, however, obtain measurements at just two wavelengths (about 650 and 950 nm). In essence, their design assumes that concentrations of methemoglobin and carboxyhemoglobin are low enough to be treated as zero.

A final important point is the acquisition of pulsed data. Usually the goal in carrying out oximetry is to determine the percentage oxygenation of the arterial blood being pumped by the heart to the systemic tissues. However, a finger contains venous blood as well as arterial, and the tissues of the finger itself can affect absorption of the wavelengths the oximeter uses. To get measures on arterial blood, oximeters take advantage of the fact that the amount of arterial blood present in a finger surges with each beat of the heart—each pulse. This explains the name pulse oximeter. By synchronizing the acquisition of primary data with the pulses, a pulse oximeter acquires information on the arterial blood and is able, through calculations, to remove confounding effects of finger tissues and venous blood present in the finger.

References

Tremper, K. K., and S. J. Barker. 1989. Pulse oximetry. Anesthesiology 70: 98–108.
(Except for calling deoxygenated hemoglobin “reduced hemoglobin,” this classic article still provides a particularly lucid explanation of the fundamental principles of pulse oximetry.)

Waterman, M. R. 1978. Spectral characterization of human hemoglobin and its derivatives. In S. Fleischer and L. Packer (eds.), Methods in Enzymology, vol. 52, pp. 456–463. Academic Press, New York.