Hemoximetry
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What state must Heme be in to bind 02?
Heme must be in the ferrous state (Fe+2) to bind 02. Not in the ferrous state = it will NOT bind heme and will be non-functional
Equation for Hgb binding O2
Hb + O2 = HbO2
Factors influencing affinity of O2 for Hgb
CO2, pH, temp, and 2,3 DPG
Functional O2 Saturation
only the functional Hgbs (oxy and deoxy)
Hemoximetry
the measurement of hemoglobin
SO2%
hemoglobin oxygen saturation
Fractional Oxygen Saturation
(%O2Hb or FO2Hb) includes the dyshemoglobins in the denominator and is written as:
When are functional and fractional O2 saturations the same?
Only when the concentrations of carboxy- and methemoglobin are zero
What happens in the presence of lethal levels of COHb?
The “functional” saturation can be normal at 98% and the “fractional” will reflect dangerously diminished oxygen-carrying capacity of the blood.
Hemoximeters are used to measure:
1) total Hb concentrations 2) O2 sat of blood
Complex (multi wave) hemoximeters measure:
1) Carboxyhemoglobin (HbCO) 2) Methemoglobin (MetHb)
Carboxyhemoglobin
HbCO - carbon monoxide bound to Hgb
Methemoglobin (MetHb)
Ferric (Fe+3) hemoglobin
Spectra of OxyHgb and DeoxyHgb from 500 to 900 nm
Which form of hemoglobin is least read and which is the reddest in the absorption spectra?
Least red - reduced hemoglobin Most red - caboxyhemoglobin (cherry red!)
Basics of Hemoximeter Operation
1) blood aspirated into instrument, hemolyzed, and placed in a light cell 2) hemoximeter measures the absorbance of light of the lubstance in the light cell (cuvette) using the principles of spectrometry
What is the visible absorbance of blood due to?
Most of the visible absorbance of blood is due to the various forms of Hgb. The remaining absorbance (plasma) is ow and ignored.
Beer-Lambert Law
For any compound, absorbance (A) is porportional to the thickness of the cuvette (light path L) and the concentration (c) of the compound.
molar extinction coefficient
a tany wavelenght the light absorbed by a particular compound is porportional to the constant molar extinction coefficient (E). Absorption spectrums for Hgb, deoxyHgb, and Carboxy Hgb are constants so we can use this to calculate the proportion of Hgb.
How many wavelengths needed to measure single substances such as Hgb?
For pure single substances like Hgb, only one wavelength is needed to measure it. However, blood has more than one form of Hgb!
How can you measure multiple Hgb forms?
By making measurements are more than 2 wavelengths and knowing the extinction coefficients for each form of Hgb at those wavelengths and knowing the ratios fo absorbances for the pure forms.
How do you measure two forms of Hgb?
by using two wavelengths (pulse oximeters measure only two wavelengths) Simple hemoximeters (Radiometer OSM-2) determine only Hgb and HbO2 at 506.5 nm and 600 nm.
Ratio of absorbance of pure deoxyHgb at 506.5 and 600nm (506.5/600)
1.43 Therefore, a measured absorbance ratio of 1.43 indicates 100% deoxyHgb
Ratio fo absorbance of pure OxyHgb at 506.5 and 600nm
5 Therefore, a measured absorbance ratio of 5 indicates 100% oxyHgb