Sulfhemoglobin Formation Research Articles

Effect of watering before slaughter and the evisceration method on the quality characteristics of fattened goose liver

The aim of this study was to investigate the effect of watering before slaughter and different evisceration methods on the quality characteristics of fattened goose liver. A total of randomly selected 60 male Greylag Landaise geese (Anser anser) were used to execute the experiment which had been force-fed during their growing period. In the slaughterhouse, randomized block design was followed and the birds were randomly divided into four experimental groups and half of the them (30 birds) were given 200 ml tap water. During the slaughter bleeding kinetics was measured and after the traditional processing steps (scalding, defeathering, waxing, water cooling, clearing of wax residues, rinsing, feet removal) 50 % of the birds (30 birds) were immediately eviscerated. The other half of the carcasses were eviscerated after 8 hours of cooling. Then pH and hemoglobin content were determined and the livers were vacuum packed and stored at -18 °C for 35 days (5 weeks). During this time color measurement and microbial analysis were taken. Results showed that pH was not influenced by neither the watering nor the evisceration methods. Hemoglobin content was significantly reduced by watering (P<0.001). Cold evisceration was proved to have siginificant effect on the color parameters (P<0.001), however the impact was not immediate, the difference was first detected on day 21. According to microbiological analysis four species were identified but none of them are known to produce hydrogen sulfide (H2S) which is required to the formation of sulfhemoglobin which compound is most likely responsible for the greenish discoloration occured during the storage. In summary, it can be stated that the evisceration method had a great impact on the color of fattened goose livers and undesirable changes - e.g. surface greening - which commonly occur in case of vacuum packed and frozen stored livers could be prevented by the application of warm evisceration.

Insights into Sulfhemoglobin Detection: UV-Vis and Fluorescence Spectroscopy Correlations

The mechanisms by which drugs and several sulfur chemicals induce sulfhemoglobin formation have not yet been elucidated. However, enzymes producing hydrogen sulfide in mammalian tissues and organs suggest sulfhemoglobin and sulfmyoglobin formation mechanisms are more complex than previously hypothesized. The process involves the interaction of H2S with hemoglobin or myoglobin in the presence of O2 or H2O2 to generate sulfhemoglobin or sulfmyoglobin, respectively. Structurally, the sulfheme product chromophore is a covalent heme modification. This modification involves the incorporation of one sulfur atom within carbon atoms to form a sulfur-carbons ring moiety across the β-β double bond of heme pyrrole B, which shows a characteristic optical band around 623 nm and 618 nm for sulfhemoglobin and sulfmyoglobin, respectively. The results show a linear correlation between the sulfHb electronic charge transfer transition at 623 nm and the emission wavelength of 460 nm upon Soret excitation at 420 nm. The data show no such linear relationship for oxy-Hb or met-aquo Hb. This new approach allows us to measure from 0.02% to 13.5% sulfhemoglobin in mixtures of met-aquo hemoglobin and oxy-hemoglobin. Although additional work is needed, the results suggest that simultaneously monitoring sulfHb electronic transition at 623 nm and emission wavelength at 460 nm upon Soret excitation at 420 nm is a powerful technique to determine the percentage of sulfhemoglobin in blood. The data and techniques presented indicate that fluorescence spectroscopy coupled with UV-vis spectroscopy provides a fast and accurate method for detecting sulfhemoglobin in the blood, facilitating the diagnosis of sulfhemoglobinemia in patients.

EPR detection of sulfanyl radical during sulfhemoglobin formation – Influence of catalase

Hemoglobin in its ferryl form oxidizes hydrogen sulfide and is transformed to sulfhemoglobin, where the sulfur is inserted covalently at the heme edge. Shown here is evidence that—as previously proposed by others—this process involves oxidation of hydrogen sulfide to a sulfanyl radical detectable by spin-trapping in electron paramagnetic resonance (EPR) spectroscopy. The yields and rates of formation of sulfhemoglobin as well as of the sulfanyl radical are affected by the same factors that affect the reactivity of hemoglobin ferryl, in bovine hemoglobin and in phytoglobins as well. A freely-diffusing sulfanyl radical is thus proposed to be involved in sulfhemoglobin formation. Catalase is shown to accelerate this process due to a previously described hydrogen sulfide oxidase activity, within which EPR evidence for sulfanyl generation is shown here for the first time. The reaction of preformed ferryl with hydrogen sulfide—in absence of hydrogen peroxide—is studied by stopped-flow at several pH values and explained in light of reactivity and redox potential control.

Investigation of the Background of Greening of Fatty Goose Liver (Foie Gras) during Prolonged Frozen Storage under Vacuum

During vacuum-packaged frozen storage of fatty goose liver (“foie gras”), a yet unknown, but fully reversible (package opening and thawing), greening was found at a Hungarian processing plant. Laboratory analysis revealed hydrogen sulfide (H2S) and remnant blood in 35 livers. Microbial spoilage was under the limit of detection for H2S-producing strains. In liver homogenates and in thawing exudates, sulfhemoglobin was found and confirmed (acid-alkali reactions), with spectrophotometric analysis. Slaughtered geese are chilled to 2C (24 h) in a non-eviscerated condition to maintain liver shape. With logistic regression, the H2S (most probably from the intestinal tract) was of primary importance in the green color development. In an experimental group (n = 10) dissected after 2 h of body chilling, H2S was absent in the liver. Results indicate that intestinal tract-originated H2S is diffusing the fattened goose liver, leading to a color defect of a delicatesse, without consumer health risks. Practical Applications Hungary leads the world's “foie gras” production, and a rarely occurring production-technology-associated green color defect negatively affects product perception. Fattened, slaughtered geese are chilled to 2C for 24 h in a non-eviscerated form to keep liver intact during dissection. Based on the industrial observations and analytical results, foie gras contains sulfhemoglobin, a non-toxic, green pigment, which is sensitive towards oxidative stimuli. The color defect occurs only in frozen-stored and vacuum-packaged livers. After excluding microbiological spoilage and proving the presence of sulfhemoglobin, we suggest the application of an oxidative packaging technology, e.g., modified atmosphere. Further conditions possibly precluding the formation of sulfhemoglobin may be fresh, warm dissection (immediately after slaughter) or the improvement of the efficacy of bleeding during slaughter. Though the green pigment has no consumer health risk, its full elimination would positively affect the quality of a high-value delicatesse.

Sulfhemoglobin formation in human erythrocytes by cystalysin, an L‐cysteine desulfhydrase from Treponema denticola

Cystalysin, isolated from the oral pathogen Treponema denticola, is an L-cysteine desulfhydrase (producing pyruvate, ammonia and hydrogen sulfide from cysteine) that can modify hemoglobin and has hemolytic activity. Here, we show that enzymatic activity of recombinant cystalysin depends upon stochiometric pyridoxal phosphate. The enzyme was not functional as an L-alanine transaminase, and had a strong preference for L-cysteine over D-cysteine. Cystalysin preferred small alpha-L-amino acids as substrates or inhibitors and was far more active towards L-cysteine than towards the other standard amino acids that undergo pyridoxal phosphate-dependent beta-elimination reactions (serine, threonine, tryptophan and tyrosine). Cystalysin tolerated small modifications to the carboxylate of L-cysteine (i.e., the methyl and ethyl esters of L-cysteine were good substrates), but the smallest possible peptide with an N-terminal cysteine, L-cysteinylglycine, was a very poor substrate. These results, combined with the implicit requirement for a free amine for pyridoxal phosphate-dependent reactions, imply that cystalysin cannot catabolize cysteine residues located within peptides. Cystalysin has Michaelis-Menten kinetics towards L-cysteine, and there was little or no inhibition by ammonia, H2S, pyruvate and acetate. Human erythrocytes incubated with H2S or with cystalysin and cysteine primarily accumulated sulfhemoglobin and methemoglobin, along with minor amounts of choleglobin and protein aggregates. Erythrocytes retained the ability to reduce methemoglobin in the presence of H2S. Cystalysin could not modify hemoglobin when beta-chloroalanine was the substrate, indicating an absolute requirement for H2S production. Cystalysin appears to be an unregulated L-cysteine catabolizing enzyme, with the resulting H2S production being essential to the atypical hemolytic activity.

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.

Intoxication of trout erythrocytes from monochloramine in vitro oxidative alteration and recovery of hemoglobin and glutathione

1. 1. Erythrocytes treated with monochloramine (NH 2Cl) underwent rapid, dose-dependent oxidation of hemoglobin (Hb) to methemoglobin (MHb) and depletion of reduced glutathione (GSH). 2. 2. MHb was reduced in the presence, but not in the absence, of glucose in the medium. 3. 3. Sulfhemoglobin formation was independent of glucose availability. 4. 4. In the absence of glucose, pyruvate regenerated GSH but did not reduce MHb. 5. 5. The GSH levels were partially restored by glucose and fully restored by 2-mercaptoethanol in the erythrocyte suspension medium. 6. 6. Alkylation of GSH with N-ethylmalemide prior to treatment with NH 2Cl increased MHb formation and decreased the rate of MHb reduction.

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.

155) - Studies on sulfhemoglobin formation by various drugs [Report 2

155) - Studies on sulfhemoglobin formation by various drugs [Report 2

EFFECT OF ELEVATED GLUCOSE CONCENTRATIONS ON THE OXIDANT SENSITIVITY OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) DEFICIENT ERYTHROCYTES

Diabetic acidosis is known to trigger hemolysis in patients with G6PD deficiency. Elevated glucose concentrations activate hexonate dehydrogenase, oxidize NADPH, and hence stimulate the hexose monophosphate (HMP) shunt. The present studies investigated whether HMP shunt activation, in the presence of increased glucose concentrations, significantly increased the vulnerability of G6PD deficient RBC's to oxidant injury. In normal RBC's, both the resting and ascorbate-stimulated HMP shunt (glucose-1-14C oxidation to 14CO2) were greater in 50 than 5 mM glucose. Glutathione regeneration in diamide-treated normal RBC's was the same at 5 and 50 mM glucose. Hydroxylaminedapsone, which requires NADPH for drug activation and maximal hemoglobin oxidation, produced more methemoglobin in normal than G6PD deficient cells, but no difference was seen when the effects of 5 and 50 mM glucose were compared. Sulfhemoglobin formation in G6PD deficient RBC's incubated with cyanide and ascorbate was not significantly different in 5 or 50 mM glucose. These data indicate that NADPH-dependent reactions vital for hemoglobin protection are not affected adversely by increased glucose concentrations. Thus, it is unlikely that hyperglycemia significantly alters the reductive potential of G6PD deficient RBC's during an oxidant stress such as infection.

OXIDANT-INDUCED INJURY IN PYRUVATE KINASE (PK) DEFICIENT ERYTHROCYTES

Oxidant-induced hemolysis, the hallmark of hexose monophosphate (HMP) shunt abnormalities/ generally is not appreciated in PK deficiency. In vitro, however, some patients with PK deficiency manifest oxidant sensitivity (increased sulfhemoglobin formation) in the presence of cyanide (CN) and ascorbate (Asc). The HMP shunt (glucose- 1-14C oxidation to 14CO2) of these PK deficient RBC's was stimulated by Asc to the same extent as were normal cells. CN enhanced the Asc-induced HMP stimulation in control cells, but inhibited the Asc effect in PK deficient RBC's. ATP was decreased significantly in PK deficient RBC's incubated with CN + Asc. Therapeutic salicylate levels (2 mM) also inhibited the Asc-induced HMP stimulation in PK deficient RBC's, but not in normal cells. These experiments suggest that inhibition of mitochondrial oxidative phosphorylation in PK deficient reticulocytes deprives these cells of adequate ATP for accelerated glucose phosphorylation in the presence of an oxidant stress. Certain drugs (salicylates) or prolonged stasis in a hypoxic splenic environment presumably could inhibit oxidative phosphorylation, impair the ability of PK deficient RBC's to detoxify oxidants produced by bacteria and macrophages during infection, and thereby induce oxidant cell injury. Furthermore, this partially may explain the exacerbation of hemolysis seen in PK deficiency during infection.

A REVALUATION OF ASPIRIN (ASA) INDUCED HEMOLYSIS ASSOCIATED WITH ERYTHROCYTE GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) DEFICIENCY

ASA has been implicated as a causative hemolytic agent in some individuals with G6PD deficiency. Gentisic acid (GA), the one known oxidant metabolite of salicylate, stimulated the haxosemonophosphate (HMP) shunt of normal RBC's. This effect, however, was seen at GA concentrations greater than 2 mM (equivalent to a minimum salicylate level of 20 mM or 350 mg%). At therapeutic salicylate concentrations of 2 mM (35 mg%) and/or with 0.2 mM GA, no effect on the resting or ascorbate-stimulated HMP shunt was noted. G6PD deficient RBC's incubated with 0.2 mM GA and/or 2.0 mM salicylate manifested no significant sulfhemoglobin formation or glutathione oxidation. Noninfected, afebrile G6PD deficient Black males given ASA (50 mg/kg/day × 4 days) had no significant changes in hemoglobin, hematocrit, reticulocytes or bilirubin. These studies suggest it is unlikely that therapeutic doses of ASA would be hemolytic in Black G6PD deficient patients. Most cases of ASA-associated hemolysis more probably are related to the reason the drug is given (ie, infection) rather than the drug per se. Instead of arbitrarily excluding this useful drug from G6PD deficient individuals, a similar ASA tolerance test done under close observation would seem a more reasonable approach for detecting ASA sensitivity.

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.

Sulfhemoglobin Formation and Labile Iron in Vitro and in Vivo

Sulfhemoglobin Formation and Labile Iron in Vitro and in Vivo

Formation of Peroxide and a Reversible Oxidation-Reduction in Solutions of Sulfanilamide

In accordance with a previous concept of the mechanism of sulf-hemoglobin formation I investigated whether sulfanilamide which often causes sulfhemoglobinemia forms H2O2 in presence of molecular oxygen. Aqueous solutions of sulfanilamide were prepared in different concentrations. The phenolphthalin reagent and, as a catalyst, copper sulfate, both as described by Schales, were added. Oxygen was bubbled through the solutions. Parallel controls contained distilled water instead of sulfanilamide solution. The development of a red color resulting from the oxidation of phenolphthalin into phenolphthalein under the given conditions is specific for the presence, which implies the formation of hydrogen peroxide or labile peroxides. Concentrations of H2O2 as low as 1:10 are detectable by means of this method. In solutions containing sulfanilamide in a concentration of 250 mg per 100 cc and more the formation of H2O2 was regularly found. In lower concentrations, e. g., 100 mg per 100 cc, no convincing specific effect was detectable. From the failure to find H2O2 in the less concentrated solutions one could conclude that possibly not the sulfanilamide itself but rather some other substance accompanying the drug as an impurity might be responsible for the H2O2 formation as described above. Two facts make such a presumption improbable but do not entirely exclude it. (1) There could not be found any characteristic differences in the behavior of different preparations of sulfanilamide. (2) Neither could there be found any shift in the limit after purifying the drug by recrystallization. Thus the formation of H2O2, although under the given experimental conditions in a rather slight amount, seems to be due to the sulfanilamide itself. That makes it likely that in vivo by means of any catalyst present, H2O2 can be formed from an oxidation of sulfanilamide.