Effect of reduced glutathione on the indexes of oxidative stress and heme metabolism in liver and blood of rats under hemin chloride injection in vivo

Heme (iron-protoporphyrin IX) is involved in various cellular functions. The release of heme under hemolysis or under the damage of intracellular hemeproteins leads to its accumulation in tissues and, as a result, to the activation of free radical processes. Reduced glutathione (GSH) functions as an endogenous water-soluble antioxidant and a regulator of cells redox status, but its effect on the development of oxidative stress under hemin action in mammals remains not investigated. The aim of this work was to study the effect of hemin chloride on some hemeproteins activity and a number of prooxidant-antioxidant status indexes in rat liver and blood under GSH level modulation in vivo. White male rats weighing 170–280 g were taken for investigation. Hemin chloride and GSH were injected intraperitoneally. Blood plasma, homogenate, and postmitochondrial fraction of liver were the objects of study. Hemin chloride injection (50 mg/kg body weight) caused the increase in heme-containing products level in blood and free heme level in liver of rats, which was accompanied by the activation of free radical processes in these tissues. The accumulation of free heme in liver was proved by an increase in tryptophan 2,3-dioxygenase (TDO) holoenzyme activity and heme saturation. The pretreatment by GSH (500 mg/kg body weight) 0.5 h before hemin chloride injection normalized GSH content, but did not prevent heme accumulation, the decrease in triglycerides level and the increase in lipid hydroperoxides content in rat blood plasma under hemin action. In liver, GSH injection prevented the increase in lipid hydroperoxides and protein carbonyl derivatives concentration as well as in TDO holoenzyme activity, and decreased the degree of TDO heme saturation. All these changes occurred under GSH content increase in liver. Catalase activity in liver did not differ from the control values after hemin chloride injection as well as after glutathione and hemin coadministration. The analysis of relationship between parameters studied in this work revealed the strong positive correlation between GSH content in plasma and liver (r=0.85; p<0.001), which was consistent with literature data on the significant role of liver in supplying other tissues with reduced glutathione. A negative correlation was found between lipid peroxidation products and triglycerides content in plasma (r=–0.52; p<0.05), which indicated the participation of triglycerides unsaturated fatty acids as substrates in the peroxidation processes under hemin action. No significant correlation between GSH and hydroperoxides content, as well as between GSH and heme-containing products levels in blood plasma was revealed. Thus, the water-soluble antioxidant glutathione was not effective enough to prevent damage of lipid components in blood under hemin chloride action in the selected dose. In the liver, on the contrary, GSH injection prevented heme accumulation and oxidative stress development under hemin action, which was obviously associated with an increase in the GSH content in this organ.

In vivo effects of hemin and nitric oxide donors on parameters of heme metabolism in rat liver and serum

In vivo effects of hemin chloride (15 mg/kg body weight) and donors of nitrogen monoxide (NO) – sodium nitroprusside (SNP, 1 mg/kg) and substrate of NO-synthase L-arginine (L-Arg, 600 mg/kg) on the activity of key enzymes of heme synthesis (5-aminolevulinate synthase, ALAS) and heme degradation (heme oxygenase, HO), on the free heme level in liver and on the content of heme in blood serum of rats were studied. NO donors were administered alone or 30 min before hemin chloride injection. The level of free heme in liver was estimated by the ratio of holoenzyme and total tryptophan 2,3-dioxygenase (TDO) activities. Two hours after hemin chloride administration a significant increase in the level of heme-containing products and lipid peroxidation products (TBARS) was found in blood serum. These changes were accompanied by decrease in ALAS activity and by increase in holoenzyme activity and heme saturation of TDO, which was the result of free heme accumulation in liver. 24 hrs after administration of hemin chloride the content of heme in serum returned to normal level, while level of TBARS remained elevated. 24 hrs after hemin action a significant increase in the activities of HO and ALAS was observed in liver, while the degree of TDO heme saturation decreased, indicating the prevalence of heme degradation over its synthesis. Both NO donors did not affect the accumulation of heme in serum and liver first hours after hemin action. However, the specific features of SNP and L-Arg effects on the key enzyme of heme synthesis in liver and the TBARS level in serum were revealed. L-Arg, unlike SNP, prevented the accumulation of TBARS in serum, but did not prevent a decrease in ALAS activity 2 hrs after hemin chloride injection. The treatment by SNP itself caused an increase in TBARS level in serum, an increase in TDO activity and a decrease in ALAS activity in liver 2 hrs after action. Heme content in serum positively correlated with holoenzyme activity and heme saturation of TDO in liver. The pretreatment with NO donors did not affect the increase in HO activity, however, it blocked the induction of ALAS, a decrease in holoenzyme activity and heme saturation of TDO 24 hrs after the administration of hemin chloride. Thus, both SNP and Arg prevented a decrease in free heme level in liver, which might be due to heme nitrosylation in the presence of NO donors and, as a result, its slower degradation in the heme oxygenase reaction.

MP4CO, a Pegylated Hemoglobin Saturated with Carbon Monoxide, Inhibits Microvascular Stasis in Transgenic Sickle Mice Through Heme Oxygenase-1.

Abstract 2112 Our laboratory has shown in murine models of sickle cell disease (SCD) that intravascular heme promotes oxidative stress, inflammation and microvascular stasis through toll-like receptor-4 (TLR4) signaling. Furthermore, the heme degrading enzyme, heme-oxygenase-1 (HO-1) and its by-products biliverdin and carbon monoxide (CO), inhibit these effects. CO may induce salutary effects in SCD to decrease vaso-occlusion by inhibiting hemoglobin S polymerization, vasodilation and anti-inflammatory actions, including induction of HO-1. MP4CO is a 4.3 g/dL solution of human hemoglobin conjugated with polyethylene glycol and saturated with CO. In the current studies, we tested the hypothesis that MP4CO would induce HO-1 in transgenic sickle mice and inhibit microvascular stasis in response to hypoxia/reoxygenation (H/R). Microvascular stasis (% non-flowing venules) was examined by intravital microscopy following 1hr of hypoxia (7% O2) and 1hr of reoxygenation (room air) in NY1DD transgenic sickle mice implanted 3 days earlier with a dorsal skin fold chamber window (DSFC). Five treatment groups of 3–6 mice were studied initially: 1) lactated Ringer's solution (LRS); 2) MP4OX (oxygen saturated MP4); 3) MP4CO; 4) oxygen-saturated stroma-free hemoglobin (SFH); 5) hemin chloride, 40 nmols/g i.p. × 3 days was administered as a positive control based on the previously-demonstrated induction of HO-1. Other than hemin chloride, all solutions (LRS, MP4CO, MP4OX, SFH) were administered i.v., 0.008 mL/g. In the first study, LRS, MP4OX, MP4CO or SFH were infused 24hr prior to H/R and in the second study the same solutions were infused 30min after hypoxia, during the reoxygenation phase of the experiment. In sickle mice treated with LRS or MP4OX 24hr prior to H/R, 25% and 22% of the venules, respectively, became static in response to H/R. However, in sickle mice treated with MP4CO 24hr prior to H/R, only 9% of the venules became static (p<0.05 MP4CO vs. LRS and MP4OX). In contrast, sickle mice treated with SFH 24hr prior to H/R developed significantly more stasis (37% stasis) than sickle mice in the other treatment groups (p<0.05). As we have previously shown, pretreatment with hemin abrogated vascular stasis in sickle mice (3% stasis, p<0.05 vs. all other groups). In additional groups of sickle mice, LRS, MP4OX, MP4CO and SFH were administered 30min after hypoxia during the reoxygenation phase. After H/R, LRS-treated animals had 26% stasis, MP4OX-treated mice had 18% stasis (p<0.05 vs. LRS) and MP4CO-treated mice had11% stasis (p<0.05 vs. LRS). Infusion of SFH 30min post-hypoxia markedly worsened stasis compared to the other treatments (44% stasis, p<0.05 vs. MP4CO, LRS and MP4OX). Infusion of MP4CO, but not LRS, MP4OX or SFH, markedly induced expression of microsomal HO-1 activity and protein, suggesting HO-1 was responsible for inhibition of stasis by MP4CO. Indeed, the HO-1 inhibitor SnPP reversed the effect of MP4CO on H/R-induced stasis in sickle mice (27% stasis with SnPP + MP4CO vs. 10% with LRS + MP4CO, p<0.05). The mechanism of HO-1 induction by MP4CO was likely due to an increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2), an important transcriptional regulator of HO-1. MP4CO induced strikingly more Nrf2 in liver nuclei than LRS, MP4OX or SFH. Induction of HO-1 by MP4CO decreased the inflammatory response in sickle mice as evidenced by a decrease in activated nuclear factor-kappa B (NF-kB) phospho-p65 in liver nuclei following H/R. We conclude that MP4CO enhances cytoprotective Nrf2-regulated proteins including HO-1 resulting in decreased NF-kB activation, inflammation and microvascular stasis in transgenic SCD mice. CO delivery via MP4CO may be beneficial in patients with sickle cell anemia. Disclosures: Belcher: Sangart Inc: Research Funding. Chen:Sangart Inc: Research Funding. Young:Sangart Inc: Employment. Burhop:Sangart Inc: Employment. Vercellotti:Sangart Inc: Consultancy, Research Funding.

Synthesis of novel porphyrin and chlorin phosphonic acids and their immobilization on metal oxides

In this study an easy and flexible access to porphyrin and chlorin phosphonic acids is presented. Novel types of phosphonic acid terminated porphyrins and chlorins were synthesized starting from commercially available red blood pigment hemin chloride. Phosphonic acid groups were linked to the porphyrinoids by amide coupling via appropriate spacer moieties. Self-assembled monolayers of the synthesized phosphonates on mesoporous TiO2 electrodes of approximately 3 μm thickness were formed. Surface concentrations in a range of 1 to 4 × 10-8 mol.cm-2 could be determined by UV-vis spectroscopy.

Erythrocyte protoporphyrin/heme ratio in the assessment of iron status.

The protoporphyrinemia of iron deficiency is well recognized. Clinically, information on the protoporphyrin/heme molar ratio in whole blood offers certain advantages over protoporphyrin measurement alone. A procedure for determining this ratio is reported. Protoporphyrin is extracted, solubilized, and measured fluorometrically. Heme (as hemin chloride) is precipitated with the blood proteins, the precipitate is dissolved in an alkaline/pyridine solvent, and the resulting bispyridine ferriprotoporphyrin is measured spectrophotometrically. The molar ratio of these two metabolites correlates well with values for plasma ferritin, plasma iron, transferrin saturation, hemoglobin, and hematocrit. In some cases the ratio increases detectably while the other variables, especially hematocrit and hemoglobin, remain normal. Evidently it is a more sensitive index to iron status. For healthy men and women, the mean ratio is 16.0 (SD, 5.3). The mean + 3 SD, or a ratio of 32, is distinctly abnormal, as shown by a confirmatory test. We validated the test by surveying routine blood specimens obtained from several population groups.