Sulfhemoglobin under the spotlight - Detection and characterization of SHb and HbFeIII-SH.

Sulfhemoglobinemia is an incurable disease caused by an overdose of sulfur-containing drugs with oxidizing properties. Its diagnosis remains hindered due to the similarity of symptoms to other pathological state - methemoglobinemia, as well as contradictory information on the structure and characteristics of sulfhemoglobin. Herein, we present sulfhemoglobinemia model on living functional human erythrocytes, designed to recreate processes which could take place in a patient body in order to complement missing information and highlight distinctiveness of two hemoglobin (Hb) adducts formed after interaction with sulfur donors. Employed techniques, UV-Vis absorption, Raman, Fourier transformed infrared (FT-IR) and electronic circular dichroism (ECD) spectroscopies, allowed to distinguish and characterize Hb adduct with sulfur atom bounded directly to the iron ion (HbFeIII-SH), and irreversibly connected to the porphyrin ring (SHb - sulfhemoglobin). Presented herein results provided also new evidence on formation of both these hemoglobin adducts inside functional erythrocytes under oxidative conditions and during sulfur-containing drug presence, what can be further translated into future physiological studies. Moreover, we found that sulfur attachment to the porphyrin ring altered Hb structure and lead to changes in protein packing inside RBCs, eventually. Interestingly, measurement of blood drop smear by Raman spectroscopy occurred the most accurate method to differentiate HbFeIII-SH and SHb, indicating potential of this technique in sulfhemoglobinemia diagnosis.

Secondary structure alterations of RBC assessed by FTIR-ATR in correlation to 2,3-DPG levels in ApoE/LDLR-/- Mice.

In the present study, we characterized the secondary structure alterations of intact red blood cells (RBCs) cytosol with special attention to the sex-related alterations in 8- and 24-week-old female and male ApoE/LDLR-/- mice, compared to age-matched female and male C57BL/6J control animals. Results were obtained with previously established methodology based on Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR). Additionally, we evaluated 2,3-DPG levels in the RBCs and showed its potential link to the hemoglobin (Hb) secondary structure alterations. Considering Hb structure alterations probed by FTIR-ATR, the ratio of turns to α-helices in 8-week-old ApoE/LDLR-/- mice suggested more pronounced secondary structure alterations within the RBCs than in the age-matched control. Sex-related differences were observed solely in 24-week-old male ApoE/LDLR-/- mice, which showed statistically significant increase in the secondary structure alterations compared to 24-week-old female ApoE/LDLR-/- mice. Similar to the secondary structure alterations, no sex-related differences were observed in the levels of 2,3-DPG in RBCs, except for 24-week-old male ApoE/LDLR-/- mice, which showed significantly higher levels compared to the age-matched female ApoE/LDLR-/- mice. Considering the age-related alterations, we observed significant increases in the intracellular 2,3-DPG of RBCs with animals' age in all studied groups, except for female ApoE/LDLR-/- mice, where a significant difference was not reported. This suggests the clear correlation between secondary structure of Hb alterations and 2,3-DPG levels for male and female murine RBC and proves a higher resistance of older female RBCs to the secondary structure changes with progression of atherosclerosis. Moreover, it may be concluded that higher 2,3-DPG levels in RBCs occurred in response to the secondary structure alterations of Hb in ApoE/LDLR-/- mice.

Prostacyclin Analogues Inhibit Platelet Reactivity, Extracellular Vesicle Release and Thrombus Formation in Patients with Pulmonary Arterial Hypertension.

(1) Background: Prostacyclin analogues (epoprostenol, treprostinil, and iloprost) induce vasodilation in pulmonary arterial hypertension (PAH) but also inhibit platelet function. (2) Objectives: We assessed platelet function in PAH patients treated with prostacyclin analogues and not receiving prostacyclin analogues. (3) Methods: Venous blood was collected from 42 patients treated with prostacyclin analogues (49.5 ± 15.9 years, 81% female) and 38 patients not receiving prostacyclin analogues (55.5 ± 15.6 years, 74% female). Platelet reactivity was analyzed by impedance aggregometry using arachidonic acid (AA; 0.5 mM), adenosine diphosphate (ADP; 6.5 µM), and thrombin receptor-activating peptide (TRAP; 32 µM) as agonists. In a subset of patients, concentrations of extracellular vesicles (EVs) from all platelets (CD61+), activated platelets (CD61+/CD62P+), leukocytes (CD45+), and endothelial cells (CD146+) were analyzed by flow cytometry. Platelet-rich thrombus formation was measured using a whole blood perfusion system. (4) Results: Compared to controls, PAH patients treated with prostacyclin analogues had lower platelet reactivity in response to AA and ADP (p = 0.01 for both), lower concentrations of platelet and leukocyte EVs (p ≤ 0.04), delayed thrombus formation (p ≤ 0.003), and decreased thrombus size (p = 0.008). Epoprostenol did not affect platelet reactivity but decreased the concentrations of platelet and leukocyte EVs (p ≤ 0.04). Treprostinil decreased platelet reactivity in response to AA and ADP (p ≤ 0.02) but had no effect on the concentrations of EVs. All prostacyclin analogues delayed thrombus formation and decreased thrombus size (p ≤ 0.04). (5) Conclusions: PAH patients treated with prostacyclin analogues had impaired platelet reactivity, EV release, and thrombus formation, compared to patients not receiving prostacyclin analogues.

Surgery After Surgery for Vestibular Schwannoma: A Case Series.

OBJECTIVE: We retrospectively evaluated the oncological and functional effectiveness of revision surgery for recurrent or remnant vestibular schwannoma (rVS). METHODS: We included 29 consecutive patients with unilateral hearing loss (16 women; mean age: 42.2 years) that underwent surgery for rVS. Previous surgeries included gross total resections (GTRs, n=11) or subtotal resections (n=18); mean times to recurrence were 9.45 and 4.15 years, respectively. House-Brackmann (HB) grading of facial nerve (FN) weakness (grades II-IV) indicated that 22 (75.9%) patients had deep, long-lasting FN paresis (HB grades: IV-VI). The mean recurrent tumor size was 23.3 mm (range: 6 to 51). Seven patients had neurofibromatosis type 2. RESULTS: All patients received revision GTRs. Fourteen small- to medium-sized tumors located at the bottom of the internal acoustic canal required the translabyrinthine approach (TLA); 12 large and small tumors, predominantly in the cerebellopontine angle, required the retrosigmoid approach (RSA); and 2 required both TLA and RSA. One tumor that progressed to the petrous apex required the middle fossa approach. Fifteen patients underwent facial neurorrhaphy. Of these, 11 received hemihypoglossal-facial neurorrhaphies (HHFNs); nine with simultaneous revision surgery. In follow-up, 10 patients (34.48%) experienced persistent deep FN paresis (HB grades IV-VI). After HHFN, all patients improved from HB grade VI to III (n=10) or IV (n=1). No tumors recurred during follow-up (mean, 3.46 years). CONCLUSIONS: Aggressive microsurgical rVS treatment combined with FN reconstruction provided durable oncological and neurological effects. Surgery was a reasonable alternative to radiosurgery, particularly in facial neurorrhaphy, where it provided a one-step treatment.