Assessment of absorbed dose of gamma rays using the simultaneous determination of inactive hemoglobin derivatives as a biological dosimeter

Abstract

Biological dosimetry based on sulfhemoglobin (SHb), methemoglobin (MetHb), and carboxyhemoglobin (HbCO) levels was evaluated. SHb, MetHb and HbCO levels were estimated in erythrocytes of mice irradiated by γ rays from a ⁶⁰Co source using the method of multi-component spectrophotometric analysis developed recently. In this method, absorption measurements of diluted aqueous Hb-solution were made at λ = 500, 569, 577 and 620 nm, and using the mathematical formulas based on multi-component spectrophotometric analysis and the mathematical Gaussian elimination method for matrix calculation, the concentrations of various Hb-derivatives and total Hb in mice blood were estimated. The dose range of γ rays was from 0.5 to 8 Gy and the dose rate was 0.5 Gy min⁻¹. Among all Hb-derivatives, MetHb, SHb and HbCO demonstrated an unambiguous dose-dependent response. For SHb and MetHb, the detection limits were about 0.5 Gy and 1 Gy, respectively. After irradiation, high levels of MetHb, SHb and HbCO persisted for at least 10 days, and the maximal increase of MetHb, SHb and HbCO occurred up to 24 h following γ irradiation. The use of this “MetHb + SHb + HbCO”-derivatives-based absorbed dose relationship showed a high accuracy. It is concluded that simultaneous determination of MetHb, SHb and HbCO, by multi-component spectrophotometry provides a quick, simple, sensitive, accurate, stable and inexpensive biological indicator for the early assessment of the absorbed dose in mice.

Appendix

The millimolar concentrations of Hb-derivatives (SHb, MetHb, HbCO and HbO₂) in diluted Hb-solution were calculated from the absorbance at 500, 569, 577 and 620 nm measured in the present study, and the millimolar absorbances determined previously (van Kampen and Zijlstra 1983). These millimolar absorbances were substituted into four linear equations (Eqs. 9–13) of the type described by the theory of multi-component spectrophotometric analysis, with the four unknown concentrations of Hb-derivatives ($C_{{\rm HbO}_2}$C_HbO2, C_HbCO, C_MetHb and C_SHb).

$$A_{500} = 7.2 C_{\text{SHb}} + 9.04 C_{\text{MetHb}} + 5.35 C_{\text{HbCO}} + 5.05 C_{{{\text{HbO}}_{ 2} }}$$

(9)

$$A_{569} = 8.1 C_{\text{SHb}} + 4.1 C_{\text{MetHb}} + 14.27 C_{\text{HbCO}} + 11.27 C_{{{\text{HbO}}_{ 2} }}$$

(10)

$$A_{577} = 8.1 C_{\text{SHb}} + 4.1 C_{\text{MetHb}} + 10 C_{\text{HbCO}} + 15.37 C_{{{\text{HbO}}_{ 2} }}$$

(11)

$$A_{620} = 20.8 C_{\text{SHb}} + 3.35 C_{\text{MetHb}} + 0.33 C_{\text{HbCO}} + 0.24 C_{{{\text{HbO}}_{ 2} }} ,$$

(12)

where, A₅₀₀, A₅₆₉, A₅₇₇ and A₆₂₀ are the absorbances at 500, 569, 577 and 620 nm, respectively. The absorption bands at these wavelengths represent the absorption maxima of MetHb, HbCO, HbO₂ and SHb, respectively. The above linear system of equations can be represented in the matrix form as:

$$\left[ {\begin{array}{*{20}c} {5.05} & {5.35} & {9.04} & {7.2} \\ {11.27} & {14.27} & {4.1} & {8.1} \\ {15.37} & {10} & {4.1} & {8.1} \\ {0.24} & {0.33} & {3.35} & {20.8} \\ \end{array} } \right]*\left[ {\begin{array}{*{20}c} {C_{{{\text{HbO}}_{ 2} }} } \\ {C_{\text{HbCO}} } \\ {C_{\text{MetHb}} } \\ {C_{\text{SHb}} } \\ \end{array} } \right] = \left[ {\begin{array}{*{20}c} {A_{500} } \\ {A_{569} } \\ {A_{577} } \\ {A_{620} } \\ \end{array} } \right].$$

This linear system of equations was solved using the Gaussian elimination method for matrix calculation (Gerald 1978), to yield the following equations (Eqs. 13–16):

$$C_{\text{SHb}} = \left( {A_{620} - 0.4423 A_{500} + 0.1066 A_{569} + 0.0516 A_{577} } \right)/18.8963$$

(13)

$$C_{\text{MetHb}} = \left( {9.0602 A_{500} - 2.6960 A_{569} - A_{577} - 35.2959 C_{SHb} } \right)/66.7508$$

(14)

$$C_{\text{HbCO}} = (A_{569} - 2.2317 A_{500} + 16.0744 C_{\text{MetHb}} + 7.9681 C_{\text{SHb}} )/2.3305$$

(15)

$$C_{{{\text{HbO}}_{ 2} }} = \left( {A_{500} - 5.35 C_{\text{HbCO}} - 9.04 C_{\text{MetHb}} - 7.2 C_{\text{SHb}} } \right)/5.05,$$

(16)

where A₅₀₀, A₅₆₉, A₅₇₇ and A₆₂₀ are the corrected absorbances measured experimentally at wavelengths 500, 569, 577 and 620 nm, respectively, for a purified, extremely dilute aqueous Hb-solution.