Methemoglobin

The trial was conducted at a single centre, the Jinja Regional Referral Hospital, in Uganda. Malaria transmission is moderate and seasonal in Jinja and the surrounding Busoga catchment area [23 (link)]. The hospital operates under severe resource constraints, and over 30 % of all admissions are due to malaria.
Children (age 1–10 years) were included if they had a positive rapid diagnostic test for both P. falciparum histidine rich protein 2 (HRP2) and lactate dehydrogenase (pLDH)(First Response Malaria Ag. (pLDH/HRP2) Combo Rapid Diagnostic Test, Premier Medical Corporation Limited, India) [24 (link)], as well as selected criteria for severe malaria: repeated seizures (two or more generalized seizures in 24 h), prostration, impaired consciousness (Blantyre Coma Score <5), respiratory distress (age-related tachypnea with sustained nasal flaring, deep breathing or sub-costal retractions). Patients were not included if they had methaemoglobin (metHb) >2 % at baseline, known chronic illness (renal, cardiac or hepatic disease, diabetes, epilepsy, cerebral palsy, or AIDS), severe malnutrition (weight-for length or height below −3 standard deviations based on WHO reference charts, or symmetrical oedema involving at least the feet). Modifications to the exclusion criteria were made with regulatory committee approval after experience with the first 20 enrolled participants. The following exclusion criteria were added: haemoglobinopathy, clinical suspicion of acute bacterial meningitis, unlikely to tolerate mask for study gas delivery, and prior quinine in the emergency department. Trial nurses or clinicians from the emergency department screened patients for eligibility using a uniform checklist and clinicians made final decisions about inclusion in the study.

Albumin–heme complex was prepared by incubating 120 μM stock solution of human albumin (Sigma; A-8763) with heme at a 1:0.9 protein to heme molar ratio to ensure that no free, uncomplexed heme remained in the preparation [22 (link)]. Human hemopexin (Sigma; H-9291) and bovine methemoglobin (MP Biomedicals; 151234) were also used. Co-incubation of apo-HmuY or apo-Tfo with hemoproteins and HmuY in apo-form with Tfo-Fe(III)heme complex was carried out in PBS (pH 7.6 and 6) at 37°C and monitored by UV-visible spectroscopy using holo-Tfo and apo-HmuY each at 10 μM [14 (link),21 (link)].

The oxidation degrees of PolyCHb sample solutions were illustrated by the MetHb content, which was measured by the published equations (Benesch et al., 1973 (link)). Calculate the hemoglobin oxidation rate constant (Kox) according to the following formula: $\mathrm{Y}=Y_{\max }\left(1-e^{-\mathrm{k}\mathrm{t}}\right)+Y_0$ Where, t represents the measurement time point, Y represents the sample’s methemoglobin content, Y_max represents the highest methemoglobin content during the experiment, Y₀ represents the initial value of methemoglobin, and K represents the sample’s oxidation rate. For oxidation reaction, the calculated K value is expressed in Koxidation, which is called K_ox for short. For the reduction reaction, the calculated K value is negative, so the reduction reaction can be understood as a “negative oxidation” reaction, and its absolute value is taken to represent the reduction rate Kreduction, or K_re for short.

For the aqueous solution H₂S/NO kinetic release studies, the i-POF was mixed with Teflon particles in a wt.% ratio of 75:25 (sample: Teflon) and converted into disc pellets with 5 mm diameter in order to avoid sample dispersion in the liquid. The prior outgassing was conducted using the same conditions as the adsorption studies. The loading of the material with H₂S/NO used the procedure already described in [34 (link),35 (link)].
The DTNB and oxyhemoglobin assays were used for the H₂S and NO release studies, respectively. These two methodologies are appropriate for these studies since they allow detecting the presence of H₂S and NO at physiological pH (pH = 7.2). The preparation of the solutions and the detailed experimental procedures are described elsewhere [34 (link),35 (link)]. Briefly, the DTNB methodology is based on the absorbance (408 nm) of the anion 5-thio-2-nitrobenzoate formed in the reaction between DTNB and H₂S [36 ]. The calibration curve and corresponding spectra of the DTNB method are presented in the (Supplementary MaterialsFigure S1). The oxyhemoglobin assay is based on the oxidation of NO to nitrate by oxyhemoglobin (HbO₂). The reaction begins with methemoglobin (metHb), which absorbs at 406 nm. A shift to 415 nm (absorbance of HbO₂) is observed, allowing the NO to be quantified [37 ].
The experiments were conducted by adding the H₂S/NO loaded pellets to the DTNB or hemoglobin solutions, respectively. The first spectrum was acquired after 2 min of the pellet addition, followed by 15 to 30 min intervals until no changes were observed in the spectra, or all the hemoglobin was consumed. The absorbance spectra were recorded using a UV/Vis spectrophotometer (Genesys 10S UV/Vis spectrophotometer from Thermo Scientific Blank, Waltham, MA, USA) at room temperature. The absorbances were measured in the 250–500 nm range for the DTNB method and 350–700 nm for the oxyhemoglobin assay. The experimental conditions used in this study are shown in Table 1.

During the experimental period, several hematological parameters such as red blood cells (RBCs), white blood cells (WBCs), hemoglobin (Hb), and mean corpuscular hemoglobin (MCH) were measured. The blood was collected through a micropipette cutting caudal peduncle of the experimental fish. To prevent blood clotting hematocrit tube was used to draw the blood [41 (link)]. The collected samples were kept in Eppendorf tubes and stored in a refrigerator (at 4 °C temp.). RBCs and WBCs count were done with the help of Neubauer Hemocytometer under light microscope following the process of Hesser [41 (link)]. Hb concentration was measured using cyan-methemoglobin following the method described by Blaxhall and Daisley [42 (link)]. RBCs, WBCs, and MCH were estimated through the following formula [43 (link)]- $\mathrm{R}\mathrm{B}\mathrm{C}\mathrm{c}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}=\frac{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{l}\mathrm{a}\mathrm{r}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{q}\mathrm{u}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\left(5\right)\times \mathrm{D}\mathrm{i}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r}\left(200\right)\times \mathrm{C}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r}\left(4000\right)}{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{m}\mathrm{a}\mathrm{l}\mathrm{l}\mathrm{s}\mathrm{q}\mathrm{u}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{d}\left(80\right)}$ $\mathrm{W}\mathrm{B}\mathrm{C}\mathrm{c}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}=\frac{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{l}\mathrm{a}\mathrm{r}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{q}\mathrm{u}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\left(1\right)\times \mathrm{D}\mathrm{i}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r}\left(40\right)}{\mathrm{V}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r}\left(0.1\right)}$ $\mathrm{M}\mathrm{C}\mathrm{H}\left(\mathrm{p}\mathrm{g}\right)=\frac{\mathrm{H}\mathrm{b}\times 10}{\mathrm{R}\mathrm{B}\mathrm{C}}$