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       Hydrogen sulfide (H2S) has various physiological and pathological effects on the human body. Sodium hydrosulfide (NaHS) is widely used as a pharmacological tool to evaluate the effects of H2S in biological experiments. Although the loss of H2S from NaHS solutions takes only a few minutes, some animal studies have used NaHS solution as an H2S donor compound in drinking water. This study examined whether 30 µM NaHS in drinking water prepared in water bottles for rats and mice remains stable for at least 12-24 hours, as suggested by some authors. Prepare a solution of NaHS (30 µM) in drinking water and immediately transfer to rat/mice water bottles. Samples were taken from the tip and inside of the water bottle after 0, 1, 2, 3, 4, 5, 6, 12 and 24 hours and the sulfide content was measured using the methylene blue method. In addition, male and female rats were administered NaHS (30 μM) for two weeks, and serum sulfide concentrations were measured every other day at the end of the first and second weeks. In a sample taken from the tip of a water bottle, the NaHS solution was unstable, dropping 72% and 75% after 12 and 24 hours, respectively; In samples taken from water bottles, the reduction in NaHS content was negligible up to 2 hours, but the reduction was 47% and 72% after 12 and 24 hours, respectively; Administration of NaHS had no effect on serum sulfide levels in male and female rats. Therefore, solutions of NaHS prepared in drinking water cannot be used to donate H2S because the solution is unstable. This route of administration exposes animals to varying and lower than expected amounts of NaHS.
       The history of hydrogen sulfide (H2S) as a toxin dates back to 17001, but its possible role as an endogenously produced biological signaling molecule was reported by Abe and Kimura2 in 1996; Over the past three decades, many functions of H2S in various human systems have been elucidated1,3, leading to the recognition that H2S donor molecules may have clinical applications in the treatment or control of certain diseases3,4, see recent review by Cirino et al; .3.
       Sodium hydrosulfide (NaHS) is widely used as a pharmacological tool to assess the effects of H2S in many cell culture and animal studies5,6,7,8. However, NaHS is not an ideal H2S donor due to its rapid conversion to H2S/HS, polysulfide contamination, oxidation and volatilization in solution4,9. In many biological experiments, NaHS dissolves in water, leading to passive evaporation and loss of H2S10,11,12, spontaneous oxidation of H2S11,12,13 and photolysis14. Sulfide in the initial solution is quickly lost due to volatilization of H2S11. In an open chamber, H2S is lost approximately 5 minutes after t1/2, and its concentration decreases by approximately 13% per minute10. Although loss of H2S from NaHS solutions takes only a few minutes, some animal studies have used NaHS solutions in drinking water as a source of H2S for 1–21 weeks, replacing the NaHS-containing solution every 12–24 hours15,16,17,18 ,19,20,21,22,23,24,25,26. This practice is inconsistent with the principles of scientific research, since drug doses must be determined for their use in other species, especially humans27.
       Preclinical research in biomedicine aims to improve patient care or health. However, the results of most animal studies do not apply to humans28,29,30. One reason for translation failure is less attention to the methodological quality of animal studies30. Therefore, this study examined whether a 30 µM solution of NaHS in drinking water, prepared in water bottles for rats and mice, remained stable for 12–24 hours, as claimed or suggested in some studies.
       All experiments in the current study were confirmed by published guidelines for the care and use of laboratory animals in Iran 31 . All experiments in the current study were also reported according to ARRIVE guidelines32. The Ethics Committee of the Institute of Endocrinology, Shahid Beheshti University of Medical Sciences, confirmed and approved all experimental procedures of this study.
       Zinc acetate dihydrate (CAS: 5970-45-6) and ferric chloride anhydrous (CAS: 7705-08-0) were purchased from Biochem, Chemopahrama (Cosne Sur Loire, France). Sodium hydrosulfide hydrate (CAS: 207683-19-0) and N,N-dimethyl-p-phenylenediamine (DMPD) (CAS: 535-47-0) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Isoflurane was purchased from Piramal (Bethlehem, PA, USA). Hydrochloric acid (HCl) was purchased from Merck (Darmstadt, Germany).
       Prepare a solution of NaHS (30 µM) in drinking water and immediately transfer to rat/mice water bottles. This concentration was chosen based on many publications in which NaHS was used as a source of H2S in studies (see Discussion section). NaHS is a hydrated molecule that can contain varying amounts of water of hydration (i.e. NaHS This value is discussed in the section where we use the molecular weight of 56.06 g/mol, which corresponds to anhydrous NaHS. Water of hydration (also called water of crystallization ) is an integral water molecule in the crystal structure33. Hydrates have different physical and thermodynamic properties compared to anhydrates34.
       Before adding NaHS to drinking water, measure the pH and temperature of the solvent. Immediately transfer the NaHS solution to the rat/mouse water bottle in the animal cage. Samples were taken from the tip of the water bottle and from the inside of the bottle at 0, 1, 2, 3, 4, 5, 6, 12 and 24 hours to measure sulfide. Sulfide measurements were taken immediately after each sampling. We obtained samples from the tip of the bottle because some studies have shown that the small pore size of water bottles can minimize the evaporation of H2S15,19. This problem seems to exist for bottled solutions. However, this is not the case for the solution at the tip of a water bottle, which has a higher rate of evaporation and auto-oxidation: in fact, animals drink this water first;
       Male and female Wistar rats were used in this study. The rats were kept in polypropylene cages (2-3 rats per cage) under standard conditions (temperature 21-26°C, humidity 32-40%), 12 hours of light (from 7:00 to 19:00) and 12 hours of darkness ( 19:00). until 7 am). They have free access to running water and regular food (Khorak Dam Pars, Tehran, Iran). Age-matched (6-month-old) female (n = 10, body weight: 190–230 g) and male (n = 10, body weight: 320–370 g) Wistar rats were randomly assigned to control and NaHS groups (30 µM )-treatment group (n = 5/group). We determined the sample size using the KISS (Keep It Simple, Stupid) method, which combines past experience with power analysis35. We first conducted a pilot study in three rats and determined the mean serum total sulfide level and standard deviation (8.1 ± 0.81 μM). Next, considering a power of 80% and assuming a two-sided significance level of 5%, we determined a preliminary sample size (n = 5 based on previous literature) that corresponds to the standardized effect for the predefined sample size value provided by Festing Quantity 2.02 Calculation. experimental animals 35. Multiplying this value by the standard deviation (2.02 × 0.81) gives a predicted detectable effect size (1.6 µm) of 20%, which is acceptable. This means that n = 5 per group is sufficient to detect an average change between groups of 20%. Rats were randomly divided into control and NaSH treatment groups using the random function of Excel software (Supplementary Figure 1). Blinding was performed at the outcome level, with investigators performing biochemical measurements blind to the groups.
       Both male and female NaHS groups were treated with a 30 μM NaHS solution prepared in drinking water, and fresh solution was given every 24 hours for 2 weeks, and body weight was measured at the same time; Blood samples were collected from the tail tip of all rats every other day during the first and second weeks under isoflurane anesthesia. Blood samples were centrifuged at 3000 g for 10 min, and serum was separated and stored at −80°C for subsequent measurement of serum urea, creatinine (Cr), and total sulfide. Serum urea was measured by the enzymatic urease method and serum Cr by the photometric Jaffe method using a commercially available kit (Man Company, Tehran, Iran) and an automatic analyzer (Selectra E, serial number 0-2124, The Netherlands). Intra- and inter-assay coefficients of variation for both urea and Cr were <2.5%.
       Total sulfide content was determined in drinking water and serum containing NaHS using the methylene blue (MB) method; MB is the most commonly used method for measuring sulfide in stock solutions and biological samples11,37. The MB method can be used to estimate the total sulfide pool38 and measure inorganic sulfides in the aqueous phase as H2S, HS- and S239. In this method, sulfur is precipitated as zinc sulfide (ZnS) in the presence of zinc acetate11,38. Precipitation with zinc acetate is the most widely used method for separating sulfides from other chromophores11. ZnS was redissolved with HCl11 in a strongly acidic medium. The sulfide reacts with DMPD, catalyzed by ferric chloride (Fe3+ as an oxidizing agent) in a stoichiometric ratio of 1:2, to form the dye MB, which is detected spectrophotometrically at 670 nm40,41. The detection limit of the MB method is approximately 1 µm11.
       In the current study, 100 μL of each sample (solution or serum) was added to the tube, followed by 200 μL zinc acetate (1% w/v dissolved in distilled water), 100 μL DMPD (20 mM dissolved in distilled water); HCl 7.2 M) and 133 µl FeCl3 (30 mM in 1.2 M HCl). The mixture was incubated at 37°C in the dark for 30 min. The solution was centrifuged at 10,000 g for 10 minutes and the absorbance of the supernatant was determined at 670 nm using a microplate reader (BioTek, MQX2000R2, Winooski, VT, USA). A calibration curve of NaHS (0–100 μM) in ddH2O was used to determine the sulfide concentration (Supplementary Figure 2). All solutions used for measurements were freshly prepared. The intra- and inter-assay coefficients of variation for sulfide measurements were 2.8% and 3.4%, respectively. Using the spike-sample method, we also determined the total amount of sulfides extracted from drinking water and serum samples containing NaSH42. The recovery rates of drinking water and serum samples containing NaSH were 91 ± 1.1% (n = 6) and 93 ± 2.4% (n = 6), respectively.
       GraphPad Prism version 8.0.2 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com) was used for statistical analysis. A paired t test was used to compare the temperature and pH of the drinking water before and after NaHS addition. H2S loss in solutions containing NaHS was calculated as the percentage decrease in absorbance relative to baseline, and one-way repeated measures ANOVA followed by Dunnett’s multiple comparison test was used to assess whether this loss was statistically significant. Two-way mixed (between groups) ANOVA and Bonferroni post hoc tests were used to compare changes in body weight, serum urea, serum Cr, and serum total sulfide over time between control and NaHS-treated sex-matched rats. Two-sided P values ​​<0.05 were considered statistically significant.
       The pH of drinking water before the addition of NaHS was 7.60 ± 0.01, after the addition of NaHS – 7.71 ± 0.03 (n = 13, p = 0.0029). The drinking water temperature was 26.5±0.2 and decreased to 26.2±0.2 after the addition of NaHS (n=13, p=0.0128). Prepare a solution of NaHS 30 µM in drinking water and store it in a water bottle. The NaHS solution is unstable and its concentration decreases over time. When samples were taken from the tip of a water bottle, a large decrease (68.0%) was observed within the first hour, and the amount of NaHS in solution decreased by 72% and 75% after 12 and 24 hours, respectively. In samples taken from water bottles, the reduction in NaHS content was negligible up to 2 hours, but the reduction was 47% and 72% after 12 and 24 hours, respectively; These data show that, regardless of sampling location, the percentage of NaHS in 30 μM solutions prepared in drinking water decreased to approximately one-quarter of the initial value after 24 h (Fig. 1).
       The stability of NaHS solution (30 µM) in drinking water is maintained in rat/mouse water bottles. After preparing the solution, take samples from the tip and inside of the water bottle. Data are means ± SE (n = 6/group). * and #, P < 0.05 vs. time 0. Image of a water bottle: tip (and its opening) and body. The tip volume is approximately 740 µl.
       The concentration of NaHS in the freshly prepared 30 μM solution was 30.3 ± 0.4 μM (range: 28.7–31.9 μM, n = 12). However, after 24 h it decreased to lower values ​​(mean: 3.0 ± 0.6 μM). As shown in Figure 2, rats were not exposed to stable concentrations of NaHS during the study period.
       The body weight of female rats increased significantly over time (from 205.2 ± 5.2 to 213.8 ± 7.0 in the control group and from 204.0 ± 8.6 to 211.8 ± 7.5 g in rats treated with NaHS, but the administration of NaHS had no effect on body weight); (Fig. 3). The body weight of male rats increased significantly over time (from 338.6 ± 8.3 to 352.4 ± 6.0 in the control group and from 352.4 ± 5.9 to 363.2 ± 4.3 g in rats received NaHS, but the administration of NaHS did not affect body weight); (Fig. 3).
       Changes in body weight of female and male rats after administration of NaHS (30 μM). Data represent means ± SEM and were compared using two-way mixed (within groups) ANOVA with Bonferroni post hoc test. n = 5/gender group.
       Serum urea and Cr concentrations were comparable between control and NaSH-fed rats throughout the study. In addition, NaSH treatment had no effect on serum urea and Cr concentrations (Table 1).
       Baseline serum total sulfide concentrations were comparable in control and NaHS-treated male rats (8.1 ± 0.5 vs. 9.3 ± 0.2 μM) and female rats (9.1 ± 1.0 vs. 6.1 ± 1.1 µM). Administration of NaHS for more than 14 days had no effect on serum total sulfide levels in male and female rats (Fig. 4).
       Change in serum total sulfide concentration after administration of NaHS (30 μM) to female and male rats. Data represent means ± SEM and were compared using two-way mixed (within groups) ANOVA with Bonferroni post hoc test. n = 5/gender group.
       The main findings of this study were that drinking water containing NaHS was unstable, with only approximately one-quarter of the original total sulfides being detected after 24 hours of sampling the tips and interiors of rat and mouse water bottles. In addition, rats were not exposed to steady-state concentrations of NaHS due to loss of H2S in the NaHS solution, and administration of NaHS to drinking water had no effect on body weight, serum urea and Cr levels, or serum total sulfide levels.
       In this study, the rate of H2S loss from a 30 µM NaHS solution prepared in drinking water was approximately 3% per hour. The hourly sulfide concentration was reported to decrease by 7% over time in a buffer solution (sodium sulfide, 100 μM in 10 mM PBS, pH = 7.4) for 8 hours. We previously defended intraperitoneal injection of NaHS by reporting that the rate of sulfide loss from drinking water containing NaHS in a 54 μM solution was approximately 2.3% per hour (4%/hour for the first 12 hours and approximately 1.4%/hour after that) after preparation of the second 12 hours)8. Early studies emphasized the constant loss of H2S from NaHS solutions, which mainly occurs through volatilization and oxidation. Even without bubbling, sulfide is quickly lost from the original solution due to the evaporation of H2S11. Research has shown that during the mother liquor dilution process, which takes about 30-60 seconds, about 5-10% of the H2S will be lost through evaporation6. To avoid evaporation of H2S from the solution, the researchers took a number of measures, including gentle stirring of the solution12, covering the original solution with parafilm6, and minimizing contact of the solution with air, since the rate of H2S evaporation depends on the air-liquid interface13. Spontaneous oxidation of H2S occurs mainly due to transition metal ions, especially ferric iron, which is present in water as impurities13. Oxidation of H2S leads to the formation of polysulfides (sulfur atoms linked by covalent bonds)11. To avoid its oxidation, solutions containing H2S are prepared in deoxygenated solvents44,45, and then the solutions are purged with argon or nitrogen for 20–30 min to ensure deoxygenation11,12,37,44,45,46. Diethylene triamine pentaacetic acid (DTPA) is a metal chelating agent (10–4 M) that prevents autoxidation of HS- in aerobic solutions. In the absence of DTPA, the temperature is about 3°C ​​at 25°C37,47. Oxidation. the rate is approximately 50% within a few hours. In addition, the solution should be stored on ice, protected from light, since the oxidation of 1e-sulfide is catalyzed by UV light.
       As shown in Figure 5, when NaHS is dissolved in water, it ionizes to form Na+ and HS-6, this dissociation depends on the pK1 reaction, which is related to temperature: pK1 = 3.122 + 1132/T, where T is between; 5 and Within 30°C and is expressed in degrees Kelvin (K), K = °C + 273.1548. HS- has a high pK2 level (pK2 = 19) so it produces no S2- or very little S2- at pH < 96.49. Instead, HS- acts as a base and accepts H+ from H2O molecules, which act as acids and become H2S and OH-.
       Dissolved H2S gas is formed in NaHS solution (30 µM). water-based aqueous solution; g – gas; l – liquid. All calculations assume water pH = 7.0 and water temperature = 20 °C. Created with BioRender.com.
       Despite evidence of instability of NaHS solutions, several animal studies have used NaHS solutions in drinking water as H2S donor compounds15,16,17,18,19,20,21,22,23,24,25,26 that prolong lifespan varied from 1 to 21 weeks (Table 2). In these studies, the NaHS solution was renewed every 1215, 17, 18, 24, 25 or 2419, 20, 21, 22, 23 hours. Our results showed that rats were not exposed to stable concentrations of the drug due to loss of H2S in the NaHS solution, and NaHS levels in rats’ drinking water fluctuated widely over 12 or 24 hours (see Figure 1). 2). Two studies reported that H2S levels in water remained stable over 24 hours or that H2S losses were only 2-3% over 12 hours, but they did not provide supporting data or measurement details. Two studies have shown that the small pore size of water bottles minimizes the evaporation of H2S15,19. However, our results show that this delays the loss of H2S inside the water bottle by only 2 hours rather than 12-24 hours. In both studies, we assumed no change in NaHS levels in drinking water because we did not see changes in water color, so air-induced H2S oxidation was not significant19,20; Surprisingly, this subjective method was used to assess the stability of NaHS in water rather than to measure changes in its concentration over time.
       The loss of H2S from NaHS solutions depends on pH and temperature. As observed in our study, dissolving NaHS in water results in the formation of an alkaline solution50. Once NaHS is dissolved in water, the formation of dissolved H2S gas is pH dependent 6. The lower the pH of the solution, the greater the proportion of NaHS present as H2S gas molecules and the greater the loss of sulfide from the aqueous solution11. None of these studies reported the pH of drinking water used as NaHS solvent. According to the recommendations of the World Health Organization, adopted in most countries, the pH of drinking water should be in the range of 6.5-8.551. In this pH range, the rate of spontaneous H2S oxidation increases approximately tenfold13. When NaHS is dissolved in water in this pH range, the concentration of dissolved H2S gas ranges from 1 to 22.5 µM, highlighting the importance of controlling the pH of the water before dissolving NaHS. Additionally, the temperature range reported in the above study (18-26°C) results in a change in the concentration of dissolved H2S gas in solution by about 10%, since changes in temperature change pK1, and small changes in pK1 can also cause dissolved gas concentration of H2S changes by about 10%. Has a significant effect on the proportion of dissolved gas H2S48. To top it off, some studies were longer (5 months) 22 during which greater temperature change was expected.
       All but one study21 used a 30 µM NaHS solution in drinking water. To explain the dose used (i.e. 30 µM), some authors argue that NaHS in the aqueous phase produces exactly the same concentration of H2S gas, and since the physiological range of H2S is between 10 and 100 µM, this dose is within the physiological range range. 15,16 It has also been explained that 30 µM NaHS can maintain plasma H2S levels within the physiological range, i.e. 5–300 µM19,20. Consider NaHS at 30 µM in water (pH = 7.0, T = 20 °C), which has been used in a number of studies to study the effects of H2S. We can calculate that the dissolved gas concentration of H2S is 14.7 µM, which is approximately 50% of the initial concentration of NaHS. This value is similar to previous calculations by others under the same conditions13,48.
       In our study, NaHS administration did not change body weight; this result is consistent with other studies in male mice22,23 and male rats18, however, two studies reported that NaSH restored weight loss in rats after nephrectomy24,26; reported no effect of NaSH administration on body weight15,16,17,19,20,21,25. Moreover, in our study, NaSH administration had no effect on serum urea and Cr, which is consistent with another report25.
       This study showed that adding NaHS to drinking water for 2 weeks did not affect serum total sulfide concentrations in male and female rats. This finding is consistent with the results obtained by Sen et al.16 where treatment with 30 µM NaHS in drinking water for 8 weeks had no effect on plasma sulfide levels in control rats, however they reported that this intervention restored the renal reduction in H2S levels in plasma; in ablated mice. Lee et al also reported that treatment with 30 μM NaHS in drinking water for 5 months increased plasma free sulfide levels by approximately 26% in aged mice22. Other studies have reported no changes in circulating sulfides following the addition of NaHS to drinking water.
       Seven studies reported that they used Sigma’s NaHS15,16,19,20,21,22,23 but did not provide additional information about the water of hydration, and five studies did not mention the source of NaHS used in their formulations17,18. 24, 25, 26. NaHS is a hydrated molecule and can have a variable water of hydration, which affects the amount of NaHS required to prepare a solution of a given molar concentration. For example, in our study it was NaHS 1.3 H2O. Thus, actual NaHS concentrations in these studies may be lower than reported.
       “How can such a short-lived compound have such a long-lasting effect?” This question was asked by Pozsgai et al 21 who assessed the effect of NaHS on colitis in mice. These authors hope that future studies will answer this question and suggest that in addition to H2S and disulfides that mediate the effect of NaHS, NaHS solutions may also contain more stable polysulfides21. Another possibility is that very low concentrations of NaHS remaining in solution may have a beneficial effect. In fact, Olson provided evidence that the micromolar range of H2S in the blood is unphysiological and that it must be in the nanomolar range or it would not be present in the blood at all13. It is possible that HS acts through protein sulfation, a reversible post-translational modification that affects the function, stability and localization of many proteins52,53,54. In fact, approximately 10–25% of many liver proteins are hydrosulfated under physiological conditions53. Two studies confirmed the rapid degradation of NaHS19,23, but unexpectedly stated that “we controlled NaHS concentrations in drinking water by changing the drinking water daily”23. One study surprisingly stated that “NaHS is the standard H2S donor that is commonly used clinically, not H2S itself” 18 .
       Based on the above discussion, which shows that NaHS is lost from solution through volatilization, oxidation and photodecomposition, some suggestions are made to minimize the loss of H2S from solution. First, the evaporation of H2S depends on the gas-liquid interface13 and the pH of the solution11, so to minimize evaporation losses, the pore size of the water bottle can be reduced as much as possible, as previously suggested15,19, and the water can be purified; The pH level is adjusted within the upper acceptable range (i.e. 6.5–8.551) to minimize the pore size of the water bottle. Second, spontaneous oxidation of H2S occurs as a result of exposure to oxygen and the presence of transition metal ions in drinking water13, so deoxygenation of drinking water using argon or nitrogen44,45 and the use of metal chelators37,47 can reduce sulfide oxidation. Third, to prevent H2S photolysis, water bottles can be wrapped in aluminum foil; this approach is also applicable to photosensitive materials such as streptozotocin 55. Finally, inorganic sulfide salts (NaHS, Na2S and CaS) can be gavaged rather than dissolved in drinking water, as previously reported56,57,58. Studies have shown that when radioactive sodium sulfide is administered to rats, it is well absorbed and distributed in the body; almost all fabrics59. To date, most studies have administered inorganic sulfide salts intraperitoneally. However, this approach has been used in clinics for at least 60 years. On the other hand, the oral route is the most common and preferred route of administration in humans 61 . We therefore propose to evaluate the effects of H2S donors in rodents using an oral gavage.
       As a limitation, we measured sulfide levels in aqueous solutions and serum using the MB method. Sulfide measurement methods include iodometric method, spectrophotometric method, electrochemical method (potentiometric method, galvanic cell method, coulometric method and amperometric method) and chromatographic method (gas chromatography and high performance liquid chromatography), among which the spectrophotometric method is the most preferred. widely used method62. A limitation of the MB method for measuring H2S levels in biological samples is that it measures all sulfur species and not free H2S63 because it is performed under acidic conditions in which sulfur64 is extracted from biological sources. However, according to the American Public Health Association, MB is the standard method for measuring hydrogen sulfide65. Thus, this limitation does not affect our main results on the instability of solutions containing NaHS. In addition, the sulfide recovery for the NaHS-containing water and whey samples we tested was 91% and 93%, respectively. These values ​​are consistent with the previously reported range (77–92)66, indicating reasonable accuracy of the assay42. As an advantage, we used rats of both sexes in accordance with National Institutes of Health (NIH) guidelines to avoid overreliance on male-only animal studies in preclinical studies 67 and included both sexes whenever possible Sex68. Others have also highlighted this issue69,70,71.
       Thus, the results of this study indicate that NaHS solutions prepared in drinking water cannot be used for H2S donation because the solution is unstable. This route of administration exposes animals to varying and lower than expected amounts of NaHS, so the findings may not be applicable to humans;
       The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
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