Sulfhemoglobin Research Articles

Interpreting sulfhemoglobin and methemoglobin in patients with cyanosis: An overview of patients with M‐hemoglobin variants

Methemoglobin (MetHb) and sulfhemoglobin (SHb) measurements are useful in the evaluation of cyanosis. When one or both values are elevated, additional analysis is important to establish the etiology of the disorder. Methemoglobinemia occurs from acquired or hereditary causes with diverse treatment considerations, while true sulfhemoglobinemia is only acquired and treatment is restricted to toxin removal. Some toxic exposures can result in a dual increase in MetHb and SHb. Hereditary conditions, such as M-Hemoglobin variants (M-Hbs), can result in increased MetHb and/or SHb values but are clinically compensated and do not require treatment if they are cyanotic but otherwise clinically well. Herein, we report 53 hemoglobin variant cases that have associated MetHb and SHb levels measured by an adapted Evelyn-Malloy laboratory assay method. Our data indicate M-Hbs cause variable patterns of MetHb and SHb elevation in a fairly reproducible pattern for the particular variant. In particular, α globin chain M-Hbs can mimic acquired sulfhemoglobinemia due to an isolated increased SHb value. If the patient appears clinically well other than cyanosis, M-Hbs should be considered early in the evaluation process to differentiate from acquired conditions to avoid unnecessary testing and treatment regimens and prompt genetic counseling.

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

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 620nm, 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 8Gy and the dose rate was 0.5Gymin-1. Among all Hb-derivatives, MetHb, SHb and HbCO demonstrated an unambiguous dose-dependent response. For SHb and MetHb, the detection limits were about 0.5Gy and 1Gy, respectively. After irradiation, high levels of MetHb, SHb and HbCO persisted for at least 10days, and the maximal increase of MetHb, SHb and HbCO occurred up to 24h 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.

P69 Oxygen reactivity in the sulfmyoglobin formation

For many years hydrogen sulfide (H2S) has been considered a toxic gas, but recent studies have presented it as a signaling gas that is involved in many physiological reactions. However, when the human body is exposed to high H2S concentrations, a sulfur atom can bind to the heme group of hemoglobin (Hb), forming an Hb derivate called sulfhemoglobin (SHb). The presence of the Histidine amino acid residue in the E7 position and the ferryl species formed by oxygen (O2), and/or hydrogen peroxide are required for its formation. Sulfhemoglobin produces a condition called Sulfhemoglobinemia, which decreases the Hb ability of O2 transport. Our goal was to evaluate the SHb complex formation with different O2 concentrations, in order to determine a SHb mechanism when formed with O2. Myoglobin (Mb) was used for experiments because it has a similar active site structure as Hb and both bind oxygen. Since it was determined that the oxyMb complex was totally formed with a 1:6 proportion (Mb: O2), O2 concentration variations were done in 1:6, 1:83, and 1:160. The SMb complex formation was evaluated by UV–vis 300–800 nm region following characteristic 620 nm sulfheme band through a 24 h kinetic analysis. Results indicated that the O2 is involved in the process of the SMb complex formation leading to the transient deoxy specie and depending on the O2concentration, the equilibrium reaction toward the deoxy specie may change.

The impact of diflubenzuron (DFB) feeding on glycosaminoglycan and sulfhemoglobin biosynthesis in one day hatched chicks Gallus domesticus (L)

Diflubenzuron (Dimilin), an inhibitor of insect chitin synthesis, was fed to neo-hatched chicks in concentrations of 50 to 2000 ppm in the diet. The activity of the vertebrate hexosamine transferases responsible for glycosa-minoglycan (mucopolysaccharide) formation was monitored by measuring the in vivo rate of incorporation of a labeled precursor into hyaluronic acid and chondroitin sulfate of skin. No inhibition of biosynthesis was noted at any concentration; indeed the insecticide appeared to stimulate the synthesis of these compounds. After 3 weeks on the diet, chicks eating 1000 and 2000 ppm diflubenzuron showed obvious signs of cyanosis, and sulfhemoglobin was demonstrated in the blood of chicks eating more than 200 ppm. The amount of sulfhemoglobin appeared to be related to the dietary insecticide content and the highest level seen was 13% of total hemoglobin in the 2000 ppm group after 31 days. These values returned to normal within 3 weeks when diflubenzuron was removed from the diet. Key words: Pesticides, insect growth regulators, diflubenzuron, chicks, glycosaminoglycan, sulfhemoglobin, Gallus domesticus.

1H nuclear magnetic resonance study of the prosthetic group in sulfhemoglobin

The molecular and electronic structure of the modified prosthetic group of sulfhemoglobin (SHb) was investigated by 1H NMR for the low-spin ferric cyano-met and high-spin ferrous deoxy sulfhemoglobin complex. The 1H NMR resonances of the two subunits in the cyano-met SHb complex were differentiated on the basis of the differential stability toward regeneration of native subunits. The subunit origin for the two sets of resonances was established by formation of the sulfglobin protein for the isolated α-chain prior to assembling with the native β-subunit to yield a tetramer with sulfhemin in the α-subunits. The subunit peak assignments establish that it is the β-subunit of SHb which regenerates more rapidly to native protein. The hyperfine shifted sulfhemin peaks were assigned based on steady-state nuclear Overhauser effects which demonstrated that similarly hyperfine shifted peaks exhibit the same dipolar connectivities observed in the analogous sulfmyoglobin complex. Hence it is concluded that pyrrole B is the site of reaction in both hemoglobin and myoglobin. The initially formed SHb complex failed to equilibrate to yield a complex with a sulfhemin sufficiently stable to extraction as found previously for sulfmyoglobin. However, apoHb readily bound the green sulfhemin extracted from the terminal alkaline equilibration product of sulfmyoglobin. The inhibition on the equilibration to the alkaline form with the exocyclic thiolene ring is attributed to the interaction with Val FG5. The observations of the same dipolar connectivities among similarly hyperfine shifted peaks in the directly prepared and reconstituted SHb complexes further support the same structure for the sulfhemin in sulfmyoglobin and SHb. The strongly hyperfine shifted peaks in the deoxy form of both SHb complexes were found very similar to those of the analogous sulfmyoglobin complexes. The proximal His labile ring proton signal appears to experience a 5- to 10-ppm decrease upon conversion of a native globin to sulfglobin. This attenuation may provide a probe for differentiating chlorins and hemins in globin pockets.

Performance of an automated six-wavelength photometer (Radiometer OSM3) for routine measurement of hemoglobin derivatives.

A new instrument for spectrophotometric determination of oxygen saturation (SO2) and the fractions of carboxyhemoglobin (FHbCO) and methemoglobin (FHi), the Hemoximeter OSM3 (Radiometer), was tested by comparative measurements with a multiwavelength method. We found that it gave reliable results for freshly drawn samples from patients. When samples with artificially induced high proportions of HbCO or Hi, or both, were introduced, results were still sufficiently accurate. The presence of sulfhemoglobin (SHb) seriously interfered with the measurement of SO2, FHbCO, and FHi, but the instrument reliably indicated its presence. Because SHb is rarely encountered, a reliable warning for its presence is adequate for clinical practice. Using the instrument in its fetal mode, we obtained accurate results for blood samples from newborns.

Diode-array spectrophotometry for simultaneous measurement of hemoglobin pigments

A prototype oxygen saturation meter was used to measure the concentrations of deoxygenated hemoglobin (rHb), oxyhemoglobin (HbO 2), carboxyhemoglobin (HbCO), methemoglobin (MetHb), and Sulfhemoglobin (SHb) in 35 μl blood. Simultaneous absorbance measurements at 535, 560, 577, 622, 636, and 670 nm permitted the composition of any hemoglobin pigment mixture to be determined more accurately, precisely and easily than before. The inclusion of 670 nm, where the hemoglobin pigments have low absorption coefficients, allowed correction for turbidity.

Green pigments in epidermal blisters of decomposed cadavers

A spectrophotometrical method for discrimination between sulfhemoglobin (SHb) and choleglobin was applied to analyses of the green pigments in epidermal blisters of decomposed cadavers. SHb appeared to be a predominant constituent of the pigments, although significant amounts of choleglobin, one third to one sixth of SHb, were also demonstrated. The present results indicate that the greenish discoloration of decomposed cadavers is due to formation of a mixture of SHb, choleglobin and possibly other denatured hemoglobin derivatives.

Studies on sulfhemoglobin formation by various drugs (5) The mechanism of sulfhemoglobin formation and species differences in methemoglobin and sulfhemoglobin formation.

The mechanism of sulfhemoglobin (SHb) formation and species differences in the formation of SHb and methemoglobin (MHb) were investigated. Erythrocytes or hemolysates were incubated with phenylhydroxylamine (PHA), aniline or its monochloro derivatives in the presence or absence of liver microsomes from various species (mouse, rat, guinea pig, rabbit, cat, dog and monkey). It was confirmed that SHb was produced by the N-hydroxy metabolites of aniline and its derivatives and was not induced via MHb. There was a relationship in SHb or MHb formation between the chemical structure of aniline derivatives and their N-hydroxy metabolite production activities in the animals (in vivo and in vitro). Species differences in the production rates of N-hydroxy metabolites from aniline and its derivatives by liver microsomes (as well as in the susceptibility of erythrocytes to N-hydroxy compounds) in vitro were not reflected in species differences in MHb and SHb formation in vivo.

STUDIES ON SULFHEMOGLOBIN FORMATION BY VARIOUS DRUGS (5) THE MECHANISM OF SULFHEMOGLOBIN FORMATION AND SPECIES DIFFERENCES IN METHEMOGLOBIN AND SULFHEMOGLOBIN FORMATION

The mechanism of sulfhemoglobin (SHb) formation and species differences in the formation of SHb and methemoglobin (MHb) were investigated. Erythrocytes or hemolysates were incubated with phenyl-hydroxylamine (PHA), aniline or its monochloro derivatives in the presence or absence of liver microsomes from various species (mouse, rat, guinea pig, rabbit, cat, dog and monkey). It was confirmed that SHb was produced by the N-hydroxy metabolites of aniline and its derivatives and was not induced via MHb. There was a relationship in SHb or MHb formation between the chemical structure of aniline derivatives and their N-hydroxy metabolite production activities in the animals (in vivo and in vitro). Species differences in the production rates of N-hydroxy metabolites from aniline and its derivatives by liver microsomes (as well as in the susceptibility of erythrocytes to N-hydroxy compounds) in vitro were not reflected in species differences in MHb and SHb formation in vivo.

Sulfhaemoglobin. Absorption spectrum, millimolar extinction coefficient at λ = 620 nm, and interference with the determination of haemiglobin and of haemiglobincyanide

The spectrophotometric properties of sulfhaemoglobin (SHb) and some derivatives were investigated using an improved technique for measuring the SHb fraction induced in human blood samples. The millimolar extinction co-efficient of SHb at λ = 620 nm was found to be 20.8 (S.D. 1.48; S.E. = 0.44; n = 11). In addition it was demonstrated that the spectral changes occurring in SHb containing haemoglobin solutions upon the addition of KCN, K 3Fe(CN) 6 and K 3Fe(CN) 6 + KCN invalidate the KCN addition method for the determination of haemiglobin. The influence of clinically occurring SHb fractions on the internationally standardized total haemoglobin determination were shown to be insignificant.

Studies of sulfhemoglobin formation by various drugs (3) (author's transl)

Sulfhemoglobin (SHb) and methemoglobin (MHb) formations by various compounds were examined by single and three consecutive intraperitoneal administrations to mice. With a single administration, methemoglobinemia was induced by diphenylamine (DPA), 1-naphthylamine (NA), phenylnaphthylamine (PNA), N-(1-naphthyl) anthranilic acid (N1) and N-(1'-naphthyl)-2-aminophenylacetic acid (IG240), and was not observed with phenylanthranilic acid (PAA), N-benzoyl-N'-phenyl-2, 6-diaminobenzoic acid (BPAA), flufenamic acid (FA), mefenamic acid (MFA), N-(2-6-dichlorophenyl) anthranilic acid (CPAA), N-(2', 6'-dichlorophenyl)-2-aminophenylacetic acid (CPPA), nine derivatives of N1 and two derivatives of IG240. On the other hand, sulfhemoglobinemia, with a single administration, was found to be induced by NA. Furthermore, with three consecutive administrations, such was induced by DPA, PAA, BPAA and FA even though SHb was not demonstrated with a single administration, and was not observed with MF, CPAA, CPPA and various derivatives of N1 and IG240.

Studies on sulfhemoglobin formation by various drugs (2) (author's transl)

Sulfhemoglobin (SHb) and methemoglobin (MHb) formations by various aniline erivatives were examined by a single and three consecutive intraperitoneal administrations to mice. It was found that with a single administraion, methemoglobinemia was induced by aniline, 2-methylaniline, 3-methylaniline, 4-methylaniline, 3-trifluoromethylaniline (3-TFMA), 4-trifluoromethylaniline (4-TFMA), 2, 3-dichloroaniline, 2-aminopehnol, acetanilide (AA), 2-hydroxyacetanilide, N-methylaniline (NMA), N,N-dimethylaniline (NDMA), N-ethylaniline (NEA) and N,N-diethylaniline, and was not observed with 2, 3-dimethylanilnie, 2-trifluoromethylaniline, 3-aminophenol, 4-aminophenol, 2. 6-dichloroaniline, 3-hydroxyacetanilide (3-HAA), 4-hydroxyacetanilide (4-HAA), 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid and 4-chloro-2-aminobenzoic acid. On the other hand, sulfhemoglobinemia which appeared much more delayed than MHb, with a single administration, was found to be induced by 3-TFMA, 4-TFMA and NMA. Furthermore, with three consecutive administrations, such was induced by AA, 3-HAA, NDMA and NEA even though SHb was not demonstrated with a single administration. Structure-SHb and MHb forming property relationships were discussed.

Determination of Hemoglobin and Its Derivatives

Publisher Summary This chapter discusses that the measurement of the total hemoglobin concentration and the determination of the hemoglobin derivatives oxyhemoglobin (HbOz), carboxyhemoglobin (HbCO), sulfhemoglobin (SHb), and hemiglobin (Hi) in blood are undoubtedly the most frequent determinations in the clinical chemical laboratory. A clinical diagnosis is often made solely on the basis of hemoglobin determinations: anemia, when values are too low; polycythemia in cases of abnormally high values. It explains that when significant variations occur in the determination of the hemoglobin concentration, it becomes difficult to give a clear-cut definition of, for example, anemia. The chapter also highlights the general requirements for the HB determination and standardization—such—as the method of choice, dilution factor, absorption spectrum, specificity, and stability.

Studies of experimental sulfhemoglobinemia.

Abstract Sulfhemoglobinemia was produced in dogs and rabbits by administration of sulfur together with acetophenetidin, TNT, or p-aminopropiophenone. The sulfhemoglobin content of the blood, the unaltered hemoglobin, red cell, reticulocyte and white cell counts, serum bilirubin content, and bile pigment excretion were followed during the formation of the sulfhemoglobin and until its disappearance after the drugs were discontinued or after transfusion. In addition, cells containing sulfhemoglobin were followed by tagging with radioiron. Formation of sulfhemoglobin occurred most rapidly on administration of p-aminopropiophenone, least rapidly with acetophenetidin. All these drugs produced hemolysis at a rate apparently unrelated to sulfhemoglobin formation. Disappearance of sulfhemoglobin was due to a random destruction process as well as senescence of the cells containing it. There is no evidence for accelerated senescence. Quantitative estimation of sulfhemoglobin in blood is discussed, and new values are given for the specific extinction coefficients of reduced carboxyhemoglobin and CO-protohemochromogen.