Interplay between up-regulation of cytochrome-c-oxidase and hemoglobin oxygenation induced by near-infrared laser.

Photobiomodulation, also known as low-level laser/light therapy (LLLT), refers to the use of red-to-near-infrared light to stimulate cellular functions for physiological or clinical benefits. The mechanism of LLLT is assumed to rely on photon absorption by cytochrome c oxidase (CCO), the terminal enzyme in the mitochondrial respiratory chain that catalyzes the reduction of oxygen for energy metabolism. In this study, we used broadband near-infrared spectroscopy (NIRS) to measure the LLLT-induced changes in CCO and hemoglobin concentrations in human forearms in vivo. Eleven healthy participants were administered with 1064-nm laser and placebo treatments on their right forearms. The spectroscopic data were analyzed and fitted with wavelength-dependent, modified Beer-Lambert Law. We found that LLLT induced significant increases of CCO concentration (Δ[CCO]) and oxygenated hemoglobin concentration (Δ[HbO]) on the treated site as the laser energy dose accumulated over time. A strong linear interplay between Δ[CCO] and Δ[HbO] was observed for the first time during LLLT, indicating a hemodynamic response of oxygen supply and blood volume closely coupled to the up-regulation of CCO induced by photobiomodulation. These results demonstrate the tremendous potential of broadband NIRS as a non-invasive, in vivo means to study mechanisms of photobiomodulation and perform treatment evaluations of LLLT.

[Parameters of oxygen-binding function of human hemoglobin modified by carbon oxide and UV-light].

The parameters of oxygen-binding function of human hemoglobin, modified by carbon oxide and UV-radiation: the pressure of half-saturation with the ligand (P50), Hill's constant (n), and arterial-venous difference of HbO2 concentration in the sample were studied. The presence of carboxyform in blood in boundaries of admissible values (lower than 10 per cents) did not noticeably influence to the oxygenation parameters. Functional properties of hemoproteid were substantially modified in case of HbCO concentration increasing from 30 up to 80 percent. It has been discovered, that the leading mechanism of protection from hemic hypoxia in case of poisoning with CO is decreasing of degree of cooperative interactions and relative affinity of hemoglobin for ligands. The stimulating influence of UV-light to the functional properties of modified with carbon oxide human hemoglobin observed in case carboxyform hemoprotein concentration in solution was lower than 10 percent. The disturbance of oxygen-binding ability of hemoglobin at the influence of higher concentrations of Hb-CO was inconvertible and was not correct with UV-radiation.

Photolyses of mammalian oxy-hemoglobin studied by nanosecond photoacoustic calorimetry.

Enthalpy and conformational volume changes in photolyses of oxy-hemoglobin (HbO(2)) of human, bovine, pig, horse and rabbit are investigated by photoacoustic calorimetry. In the experiment, a pulsed Nd:YAG laser is used as an exciting source, and a PVDF film transducer and a PZT transducer are used to detect the photoacoustic signals. Based on the time scales of the excitation and detection systems as well as the photolysis processes of HbO(2), it can be indicated that the measured enthalpy and conformational volume changes are related to slow geminate recombination and tertiary relaxation in photolyses of HbO(2), which are with the time scale of 30-40 ns and 100-150 ns, respectively. The results show that the enthalpy and conformational volume changes are different for both photolysis processes of HbO(2) and also for various mammals. The different results among the five mammals are analyzed and discussed briefly.

No effect of the hemoglobin solution HBOC-201 on the response of the rat R1H tumor to fractionated irradiation.

BACKGROUND AND PURPOSE: Tumor hypoxia is regarded as one important underlying feature of radioresistance. The authors report on an experimental approach to improve tumor response to radiation by combining fractionated irradiation with HBOC-201, an ultrapurified polymerized hemoglobin solution, which is currently used in clinical phase II/III trials as alternative oxygen carrier and proved to be highly effective in tissue oxygenation (tpO(2)). MATERIAL AND METHODS: Subcutaneously growing rhabdomyosarcoma R1H tumors of the rat were treated with either 40 Gy (2 Gy/fraction, 20 fractions in 2 weeks, ambient) followed by graded top-up doses (clamped) alone, or in combination with HBOC-201, or with HBOC-201 plus carbogen (95% O(2) + 5% CO(2)). Local tumor control (TCD50%) and growth delay were used as endpoints. In addition, the effect of HBOC-201 alone or in combination with carbogen on the tpO(2) of tumor and muscle was determined using a flexible stationary probe (Licox, GMS). RESULTS: TCD50% values of 119 Gy (95% confidence interval 103;135), 111 Gy (84;138), and 102 Gy (83;120) were determined for tumors irradiated alone, in combination with HBOC-201, and with HBOC-201 plus carbogen, respectively. Although the dose-response curves showed a slight shift to lower doses when HBOC-201 or HBOC-201 plus carbogen was added, the differences in TCD50% were not statistically significant. No effect was seen on the growth delay of recurrent tumors. HBOC-201 alone did not effect tumor or muscle tpO(2). In combination with carbogen the mean tpO(2) of muscle raised from 23.9 mmHg to 59.3 mmHg (p < 0.05), but this effect was less pronounced than the increase in tpO(2) by carbogen alone. CONCLUSION: Low-dose application of HBOC-201 does not improve the response of the rhabdomyosarcoma R1H of the rat to fractionated irradiation.

Effect of photothermal therapy on breast tumor vascular contents: noninvasive monitoring by near-infrared spectroscopy.

The goal of this study was to investigate the effect of photothermal laser irradiation on rat breast tumor (DMBA-4) vascular contents. An 805-nm diode laser was used in our experiment with a power density ranging from 0.32 to 1.27 W/cm2. The dynamic changes of oxygenated hemoglobin and total hemoglobin concentrations, delta[HbO2] and delta[Hb]total, in rat tumors during photothermal irradiation were noninvasively monitored by a near-infrared spectroscopy system. A multichannel thermal detection system was also used simultaneously to record temperatures at different locations within the tumors. Our experimental results showed that: (1) photoirradiation did have the ability to induce hyperthermic effects inside the rat breast tumors in a single exponential trend; (2) the significant changes (P < 0.005) of delta[HbO2] and delta[Hb]total in response to a low dosage of laser irradiation (0.32 W/cm2) have a single exponential increasing trend, similar to that seen in the tumor interior temperature; and (3) the increase in magnitude of delta[HbO2] is nearly two times greater than that of delta[Hb]total, suggesting that photoirradiation may enhance tumor vascular oxygenation. The last observation may be important to reveal the hidden mechanism of photoirradiation on tumors, leading to improvement of tumor treatment efficiency.

Reduction of methemoglobin via electron transfer from photoreduced flavin: restoration of O2-binding of concentrated hemoglobin solution coencapsulated in phospholipid vesicles.

Ferric methemoglobin is reduced to its ferrous form by photoirradiation either by direct photoexcitation of the heme portion to induce electron transfer from the surrounding media (Sakai at al. (2000) Biochemistry 39, 14595-14602) or by an indirect electron transfer from a photochemically reduced electron mediator such as flavin. In this research, we studied the mechanism and optimal condition that facilitates photoreduction of flavin mononucleotide (FMN) to FMNH(2) by irradiation of visible light, and the succeeding reduction of concentrated metHb in phospholipid vesicles to restore its O(2) binding ability. Visible light irradiation (435 nm) of a metHb solution containing FMN and an electron donor such as EDTA showed a significantly fast reduction to ferrous Hb with a quantum yield (Phi) of 0.17, that is higher than the method of direct photoexcitation of heme (Phi = 0.006). Electron transfer from a donor molecule to metHb via FMN was completed within 30 ns. Native-PAGE and IEF electrophoresis indicated no chemical modification of the surface of the reduced Hb. Coencapsulation of concentrated Hb solution (35 g/dL) and the FMN/EDTA system in vesicles covered with a phospholipid bilayer membrane (Hb-vesicles, HbV, diameter: 250 nm) facilitated the metHb photoreduction even under aerobic conditions, and the reduced HbV restored the reversible O(2) binding property. A concentrated HbV suspension ([Hb] = 8 g/dL) was sandwiched with two glass plates to form a liquid layer with the thickness of about 10 microm (close to capillary diameter in tissue, 5 microm), and visible light irradiation (221 mW/cm(2)) completed 100% metHb photoreduction within 20 s. The photoreduced FMNH(2) reacted with O(2) to produce H(2)O(2), which was detected by the fluorescence measurement of the reaction of H(2)O(2) and p-nitrophenylacetic acid. However, the amount of H(2)O(2) generated during the photoreduction of HbV was significantly reduced in comparison with the homogeneous Hb solution, indicating that the photoreduced FMNH(2) was effectively consumed during the metHb reduction in a highly concentrated condition inside the HbV nanoparticles.

Light absorption of (oxy-)hemoglobin assessed by spectroscopic optical coherence tomography.

The combination of optical coherence tomography and spectroscopy may allow for highly localized, quantitative measurements of tissue spectral properties. We present, for the first time to our knowledge, quantitative measurements of the absorption coefficients of phantoms and of hemoglobin and oxygenated hemoglobin with spectroscopic optical coherence tomography (SOCT). Our results suggest that SOCT will be able to provide localized, quantitative oxygenation measurements.

UVA-ketoprofen-induced hemoglobin radicals detected by immuno-spin trapping.

Ketoprofen (3-benzoyl-alpha-methylbenzeneacetic acid, KP) is a widely used nonsteroidal anti-inflammatory drug (NSAID) that causes both phototoxicity and photoallergy. Here, we investigated the formation of hemoglobin radicals, in both purified hemoglobin and red blood cells (RBC), induced by ultraviolet A (UVA)-KP by using "immuno-spin trapping," a novel approach that combines the specificity of spin trapping with the sensitivity of antigen-antibody interactions. The methemoglobin (metHb) radicals react covalently with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to form nitroxyl radical adducts that are oxidized to the corresponding nitrone adducts, which in turn are specifically recognized by antiserum against DMPO nitrone. We found that the formation of nitrone adducts in metHb depended on the UVA dose, the KP concentration and the presence of DMPO, as determined by enzyme-linked immunosorbent assay and Western blotting. Adduct formation decreased when irradiation was carried out in the presence of catalase or nitrogen, suggesting that H2O2 plays a key role in KP-UVA-induced metHb radical formation. KP in the dark did not generate metHb radical-derived nitrone adducts, whereas UVA alone resulted in the formation of metHb radical-derived nitrone adducts that increased with UVA dose from 4 to 10 J/cm2. However, KP (25 and 200 microM) plus UVA (4 and 10 J/cm2) resulted in a significant increase in the formation of metHb radical-derived nitrone adducts as compared with UVA or KP alone, indicating that KP photosensitized the production of the metHb radicals in the presence of UVA. In contrast, no metHb radical-derived nitrone adduct was detected in the absence of DMPO, even though KP and UVA were present. We also detected the hemoglobin radical formation in RBC as well as in hemolysates. The endogenous antioxidants and exogenous reduced glutathione inhibited the protein radical formation. These studies have shown that the immuno-spin-trapping technique can be used to detect radical damage in proteins as a result of photosensitizing reactions. The successful detection of protein radical formation caused by KP photosensitization could help further understand the photoallergic effect of this NSAID.

Red blood cell magnetophoresis.

The existence of unpaired electrons in the four heme groups of deoxy and methemoglobin (metHb) gives these species paramagnetic properties as contrasted to the diamagnetic character of oxyhemoglobin. Based on the measured magnetic moments of hemoglobin and its compounds, and on the relatively high hemoglobin concentration of human erythrocytes, we hypothesized that differential migration of these cells was possible if exposed to a high magnetic field. With the development of a new technology, cell tracking velocimetry, we were able to measure the migration velocity of deoxygenated and metHb-containing erythrocytes, exposed to a mean magnetic field of 1.40 T and a mean gradient of 0.131 T/mm, in a process we call cell magnetophoresis. Our results show a similar magnetophoretic mobility of 3.86 x 10(-6) mm(3) s/kg for erythrocytes with 100% deoxygenated hemoglobin and 3.66 x 10(-6) mm(3) s/kg for erythrocytes containing 100% metHb. Oxygenated erythrocytes had a magnetophoretic mobility of from -0.2 x 10(-6) mm(3) s/kg to +0.30 x 10(-6) mm(3) s/kg, indicating a significant diamagnetic component relative to the suspension medium, in agreement with previous studies on the hemoglobin magnetic susceptibility. Magnetophoresis may open up an approach to characterize and separate cells for biochemical analysis based on intrinsic and extrinsic magnetic properties of biological macromolecules.

Developments toward diagnostic breast cancer imaging using near-infrared optical measurements and fluorescent contrast agents.

The use of near-infrared (NIR) light to interrogate deep tissues has enormous potential for molecular-based imaging when coupled with NIR excitable dyes. More than a decade has now passed since the initial proposals for NIR optical tomography for breast cancer screening using time-dependent measurements of light propagation in the breast. Much accomplishment in the development of optical mammography has been demonstrated, most recently in the application of time-domain, frequency-domain, and continuous-wave measurements that depend on endogenous contrast owing to angiogenesis and increased hemoglobin absorbance for contrast. Although exciting and promising, the necessity of angiogenesis-mediated absorption contrast for diagnostic optical mammography minimizes the potential for using NIR techniques to assess sentinel lymph node staging, metastatic spread, and multifocality of breast disease, among other applications. In this review, we summarize the progress made in the development of optical mammography, and focus on the emerging work underway in the use of diagnostic contrast agents for the molecular-based, diagnostic imaging of breast.

Time-resolved fluorescence reveals two binding sites of 1,8-ANS in intact human oxyhemoglobin.

Time-resolved fluorescence of 1,8-anilinonaphthalene sulfonate (1,8-ANS) fluorescent probe bound to intact human oxyhemoglobin (HbO2) is investigated. Fluorescence emission spectra of 1,8-ANS in a potassium buffer solution (pH 7.4) of HbO2 undergo a substantial blue shift during first 6 ns after pulsed optical excitation at 337.1 nm. Nonexponential fluorescence kinetics of 1,8-ANS in the HbO2 solution are studied by the decay time distribution and conventional multiexponential analyses for a set of emission wavelength range of lambdaem = 455-600 nm. These fluorescence decays contain components with mean decay times of <0.5 ns, 3.1-5.5 ns, and 12.4-15.1 ns with spectrally-dependent relative contributions. The shortest decay component is assigned to free 1,8-ANS molecules in the bulk buffer environment, whereas the two longer decay components are assigned to two types of binding sites of 1,8-ANS in the HbO2 molecule presumably differing by polarity and accessibility to water molecules. The results represent the first experimental evidence of heterogeneous binding of 1,8-ANS to intact human oxyhemoglobin.

Nanosecond time-resolved absorption studies of human oxyhemoglobin photolysis intermediates.

The time-resolved spectra of photoproducts from ligand photodissociation of oxyhemoglobin are measured in the Soret spectral region for times from 10 ns to 320 microseconds after laser photolysis. Four processes are detected at a heme concentration of 80 microM: a 38-ns geminate recombination, a 137-ns tertiary relaxation, and two bimolecular processes for rebinding of molecular oxygen. The pseudo-first-order rate constants for rebinding to the alpha and beta subunits of hemoglobin are 3.2 x 10(4) s-1 (31 microseconds lifetime) and 9.4 x 10(4) s-1 (11 microseconds lifetime), respectively. The significance of kinetic measurements made at different heme concentrations is discussed in terms of the equilibrium compositions of hemoglobin tetramer and dimer mixtures. The rebinding rate constants for alpha and beta chains are observed to be about two times slower in the dimer than in the tetramer, a finding that appears to support the observation of quaternary enhancement in equilibrium ligand binding by hemoglobin tetramers.

[Oxygen-binding properties of human hemoglobin solutions UV-irradiated in the presence of iodoacetamide]. / Kislorodsviazyvaiushchie svoistva rastvorov gemoglobina cheloveka, UF-obluchennykh v prisutstvii iodatsetamida.

Changes of oxygen-binding properties of human hemoglobin solutions modified by the different concentrations of iodoacetamide and by this chemical agent together with UV-light in doses 151 and 755 J/m2 has been studied by means of spectroscopy. It has been shown that addition of iodoacetamide to native hemoglobin solution (12:1) results in increase of the oxygen affinity and breach of the hem-hem interaction of hemoprotein. It has been ascertained that influence of UV-radiation on hemoglobin displays in photodissociation of the tetramers to protomers.

Formation of two hydrogen bonds from the globin to the heme-linked oxygen molecule in Ascaris hemoglobin.

We have tried to find out why Ascaris hemoglobin has such an exceptionally high oxygen affinity (P50 approximately 0.004 mmHg; 1 mmHg = 133 Pa). Following Kloek et al., we have synthesized the N-terminal globin domain of Ascaris hemoglobin in Escherichia coli [Kloek, A. P., Yang, J., Mathews, F. S. & Goldberg, D. (1993) J. Biol. Chem. 268, 17669-17671]. Like Kloek et al., we found its oxygen affinity to be as high as that of native Ascaris hemoglobin. We thought that this high affinity might be due to the heme-bound oxygen molecule being stabilized by two hydrogen bonds from the globin instead of the usual one. Ascaris hemoglobin has a distal glutamine instead of the more usual histidine as one of the potential hydrogen bond donors. In addition, it contains a tyrosine at position 10 of B helix (B10) in place of the leucine generally found there in vertebrate myoglobins and hemoglobins. Following the discovery of Carver et al. that sperm whale myoglobin with the replacement of leucine B10 by phenylalanine has a raised oxygen affinity, we have replaced tyrosine B10 in the N-terminal domain of Ascaris hemoglobin by either leucine or phenylalanine [Carver, T. E., Brantley, R. E., Jr., Singleton, E. W., Arduini, R. M., Quillin, H. L., Phillips, G. N., Jr., & Olson, J. S. (1992) J. Biol. Chem. 267, 14443-14450]. Either of these replacements lowered the oxygen affinity about 100-fold, to the same level of that of human alpha-globin chains. These results are consistent with a hydrogen bond linking the tyrosine hydroxyl to the heme-linked oxygen, with a bond energy of 2.7 kcal/mol.

Oxygen effect in the radiolysis of proteins. III. Haemoglobin.

Radiolysis of haemoglobin was carried out in phosphate buffer under air, N2 or N2O and with and without ethanol. Radiation products were separated by SDS-PAGE. The loss of subunits and simultaneous aggregation and fragmentation of haemoglobin was measured, if OH-radicals were unscavenged. There was no sensitizing effect of oxygen on the degradation process. Radiation-induced fragmentation was not a random process, but produced specific fragments. The estimated molecular weights of these fragments gave further support to the assumption that the aminoacyl-proline peptide group is the preferential breaking site if OH radicals react with proteins in the presence of oxygen. In contrast with lactate dehydrogenase and bovine serum albumin such fragmentation was observed not only after aerobic radiolysis but also under anaerobic conditions. This difference must be caused by the Feporphyrin system which reacts with H2O2 under release of oxygen. If haemoglobin was irradiated under air the yield of aggregates was much lower than under N2O or N2.

In vitro and in vivo ultraviolet-induced alterations of oxy- and deoxyhemoglobin.

Ultraviolet (UV) radiation was found to convert oxyhemoglobin and deoxyhemoglobin stoichiometrically into methemoglobin and a met-like product, respectively. The peak conversion efficiency for oxyhemoglobin occurred at 285 nm and decreased by a factor of 100 by 315 nm. The peak conversion efficiency for deoxyhemoglobin occurred at 290 nm and decreased by a factor of 30 by 320 nm. The transformation of oxyhemoglobin to methemoglobin was also documented in intact erythrocytes using UV-B radiation. Finally, similar transformations were found to occur in human skin with UV-B exposure but not on all volunteers tested. These results imply that methemoglobin will be formed in vivo on solar exposure and provide evidence that UV-B photons reach the blood vessels.

[The spectral and paramagnetic properties of oxyhemoglobin solutions UV-irradiated in the presence of ascorbic acid]. / Spektral'nye i paramagnitnye svoistva rastvorov oksigemoglobina, UF-obluchennykh v prisutstvii askorbinovoi kisloty.

Absorption spectra and ESR of aqueous and aqueous/glyceric solutions of oxyhemoglobin exposed to UV radiation (250-400 nm) at 293 and 77 K in the presence of ascorbic acid have been analyzed. Vitamin C (5 x 10(-5) M) has been shown to exert a photoprotective effect with regard to oxyhemoglobin (2 x 10(-6) M) UV-irradiated with a dose of 0.86 x 10(5) J/m2 at 293 K. The photoprotective effect of ascorbic acid is also displayed after UV irradiation of frozen (77 K) aqueous/glyceric oxyhemoglobin solutions (2.53 x 10(-5) M). It is concluded that ascorbic acid can be a scavenger with respect to active UV-induced particles in protein systems, including O2-. and OH. Proposed is a mode of processes leading to UV inactivation of hemoprotein molecules.

Protection of pig epidermis against radiation-induced damage by the infusion of BW12C.

BW12C, which was developed as an agent for the treatment of sickle cell anaemia, increases the binding of oxygen to haemoglobin and hence reduces the availability of oxygen to tissues. Due to these changes in oxygen availability BW12C could act as a protector against radiation-induced injury to normal tissues. In this study the potential value of BW12C, as a radioprotector, was studied in the irradiated epidermis of the pig. The infusion of BW12C caused an instant left shift of the oxygen dissociation curve, an effect that lasted for approximately 1.5 h. This left shift in the oxygen dissociation curves increased with increasing dose of the drug. There appeared to be no long-term systemic effects produced by doses of 20-100 mg/kg of BW12C. In the first 90 min after the infusion of BW12C skin fields were irradiated with single doses of beta-rays from strontium-90 plaques. The incidence of moist desquamation was used as an endpoint for assessing the severity of the radiation response. With animals breathing approximately 70% oxygen in the anaesthetic gas mixture, the ED50 values for moist desquamation were 30-31 Gy after a dose of 30 and 50 mg/kg, and 37-38 Gy for 75 and 100 mg/kg doses of BW12C. These ED50 values were significantly higher than the value of 27.3 Gy for radiation alone. This indicated dose modification factors (DMF) with mean values of approximately 1.13 and approximately 1.40 for irradiation following the infusion of low (30-50 mg/kg) and high (75-100 mg/kg) doses of the drug, respectively. With the animals breathing air (approximately 21% of oxygen) in the 2% halothane anaesthesia gas mixture, irradiation in the presence of 30 and 50 mg/kg of BW12C resulted in ED50 values of approximately 39 Gy for moist desquamation, which was significantly higher than the value of 31.2 Gy for radiation alone. Surprisingly, a higher dose of 75 mg/kg of BW12C resulted in a lower ED50 value for moist desquamation of 34.38 Gy. Irradiation in the presence of a dose of 100 mg/kg of BW12C produced an ED50 value which was not significantly different from that for radiation alone. In the situation where animals were breathing air (approximately 21% oxygen) during irradiation a DMF of 1.14 was obtained for irradiation alone, when the results were compared with those for irradiation alone with approximately 70% oxygen in the anaesthetic gas mixture.(ABSTRACT TRUNCATED AT 400 WORDS)

[Effect of alcohols on photochemical transformations of mouse oxyhemoglobin]. / Vliianie nekotorykh spirtov na fotokhimicheskie prevrashcheniia oksigemoglobina myshei.

The spectral method was used to study the influence of such alcohols as xylene, mannitol, sorbitol and dulcitol on photochemical oxidation of mouse oxyhemoglobin molecules (6.68 X 10(-5) M). Mannitol (5.35 X 10(-4) M) was shown to exert a protective action with regard to UV-irradiated hemoproteid: the extent to which oxyhemoglobin molecules were oxidized in the presence of mannotol was lesser than that in its absence. Sorbiol or xylene (5.35 X 10(-4) M) added to an aqueous solution of hemoproteid failed to exert a photoprotective effect with respect to protein molecules.

Mössbauer effect study of gamma-irradiated human oxyhemoglobin.

Preliminary results of the Mössbauer effect study of human adult oxyhemoglobin in erythrocytes exposed to gamma-irradiation with doses of approximately 100, approximately 300 and approximately 600 kGy are presented. Mössbauer spectra measured at 87 K have been analyzed in two ways. At first, to fit these spectra we used the four components oxyhemoglobin, deoxyhemoglobin, hemochromes and non-heme Fe(III) compound which had been obtained earlier from Mössbauer spectra of X-irradiated oxyhemoglobin by Chevalier et al. (1983). However, this approximation was not satisfactory. Then a new model of spectral fitting with five components was used. These were oxyhemoglobin, deoxyhemoglobin and components marked 1, 2 and 3. Using Mössbauer hyperfine parameters of each component the valence/spin states of iron ions were determined and possible complexes were considered. The most probable compounds for components 1, 2 and 3 were hematin and/or mu-oxodimers, methemoglobin hydroxide and/or hemichromes, and the high spin Fe(III) complex, respectively. Changes of the relative areas of Mössbauer subspectra of all components (its content in samples) versus doses were evaluated and the presence of the high and low spin aquomethemoglobin was indicated.