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Development of a derivatization method for the quantification of hydrogen sulfide and its application in vascular calcification rats
Accepted Manuscript Title: Development of a derivatization method for the quantification of hydrogen sulfide and its application in vascular calcification rats Authors: Xiao-Xin Tan, Kao-Qi Lian, Xiang Li, Nan Li, Wei Wang, Wei-Jun Kang, Hong-Mei Shi PII: DOI: Reference:
S1570-0232(16)31327-7 http://dx.doi.org/doi:10.1016/j.jchromb.2017.04.023 CHROMB 20562
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
26-11-2016 28-2-2017 12-4-2017
Please cite this article as: Xiao-Xin Tan, Kao-Qi Lian, Xiang Li, Nan Li, Wei Wang, Wei-Jun Kang, Hong-Mei Shi, Development of a derivatization method for the quantification of hydrogen sulfide and its application in vascular calcification rats, Journal of Chromatography Bhttp://dx.doi.org/10.1016/j.jchromb.2017.04.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development of a derivatization method for the quantification of hydrogen sulfide and its application in vascular calcification rats Xiao-Xin Tan 1, Kao-Qi Lian 1, Xiang Li, Nan Li, Wei Wang, Wei-Jun Kang, Hong-Mei Shi * Department of Health inspection, School of Public Health, Hebei Medical University, Shijiazhuang 050017, China
Highlights:
Areliable and sensitive HPLC method for the determination of H2S was reported.
The method is sensitive enough to be applied in plasma and various tissues.
The derivatization reaction for H2S by the isomers (MMB and MBB) was first compared.
The proposed method has been applicable for studies in the VC model of rats.
It might be an useful tool for the investigation on the pathological of H2S.
Abstract: Hydrogen sulfide (H2S) plays major functional and structural roles in diverse physiological functions and the pathogenesis of a variety of disorders in biological matrices. The significance of H2S has prompted the development of sensitive and selective methods to determine its concentration in biological samples. The fluorescent reagent monobromobimane (MBB) has been widely used to measure various thiol-containing species through alkylation. MBB may prevent the oxidation of sulfide and the reaction of sulfide with several different species (such as superoxide radicals, hydrogen peroxide and peroxynitrite). An isomers of MBB, 3-(bromomethyl)-2, 6, 7-trimethyl-1H, 5H-pyrazolo [1, 2a] pyrazole-1, 5-dione (MMB), is cheaper than MBB and its use in the analysis of H2S has not
1
These authors contributed equally to this work.
*Corresponding author. Addresses: Department of sanitary inspection, School of Public
Health, Hebei Medical University, East Zhongshan Road 361, Shijiazhuang, P. R.China. E-mail: [email protected]. (H.M. Shi).
previously been reported. In the present study, we compared the derivatization reactions of hydrogen sulfide with MMB and MBB and developed a sensitive method to quantify H2S in blood. In our method, H2S was incubated in the dark with excess MMB in 0.1M Tris-HCl buffer (pH 10.1) at 50 ℃ for 120min. 50 µL aliquots of the derivatized product were analyzed using HPLC system with gradient elution of 0.1% (v/v) formic acid-acetonitrile. The limit of detection for the derivatized product was 0.03 nmol/mL. The derivatization reaction was suitable for detecting low concentrations of H2S. The derivate product is stable over time, permitting batch storage and analysis. Keywords：Hydrogen sulfide; Monobromobimane; Isomers; Derivatization; HPLC; Vascular calcification 1. Introduction
Hydrogen sulfide (H2S) is produced from a variety of sources. It is a colorless, flammable, water-soluble gas with an offensive odor. H2S is produced during amino acid metabolism by the trans-sulfuration and cysteine desulfuration pathways. H2S production is predominantly catalyzed by three tissue-specific enzymes, cystathionine β-synthase
(CBS),
cystathionine
γ-lyase
(CSE),
and
3-mercaptopyruvate
sulfurtransferase (3MST). In recent years, H2S has received increased research attention along with other gaseous mediators (such as nitric oxide and carbon monoxide), as it plays a critical role in diverse physiological functions, including neuronal, inflammatory processes, cardiovascular and endocrine processes [1-3]. Hydrogen sulfide has also been reported to participate in the pathogenesis of a variety of disorders, including portal hypertension, Alzheimer’s disease, pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD) and ulcerative colitis [4-7]. Accurately and reliably measuring the concentration of biologically free H2S in biological matrices can
provide information regarding the amounts involved in physiological and pathological responses. H2S can exist as different species, depending on the pH of the surrounding environment. In aqueous solution, free H2S is a weak acid with two acid dissociation constants [8]. In vivo, H2S exists primarily as the highly reactive hydrosulfide (HS−) anion because of the mildly basic pH. Furthermore, the oxidation of sulfide and its reaction with species such as superoxide radicals, hydrogen peroxide, and peroxynitrite, adversely affect the determination of absolute H2S in biological samples [9].It is rare to have a consensus for the concentrations of H2S associated with normal or pathological physiological processes. The unstable nature of H2S in solution makes measurement and analysis of H2S in biological matrices difficult. Several different methods have been developed for detecting H2S, including electrochemical and colorimetric methods [10-13], gas chromatography [14-15] and derivatization methods[16], using pentafluorobenzyl bromide [17] or HF-PBA [18], for example. Because of the susceptibility of sulfide to oxidation, and the reversible nature of sulfide bonds in blood and tissues, we sought to develop a simple method that quickly captures reactive sulfide without releasing chemically bound sulfur within a biological matrix, to obtain measurements that accurately quantify biologically available sulfide. The fluorescent reagent monobromobimane (MBB) has been widely used to measure various thiol-containing species through alkylation [19-20]. MBB can reacts with HS− under suitable conditions to produce sulfide-dibimane (SDB). SDB allows for the quick and accurate determination of absolute H2S present in the biological media [9, 19, and 21]. 3(bromomethyl)-2, 6, 7-trimethyl-1H, 5H-pyrazolo [1, 2-a] pyrazole-1, 5-dione (MMB) is an isomer of MBB (the structural formulas are shown in Fig.1). In this study, we found that MMB reacted readily with HS− under suitable conditions to produce sulfide-pardimane (SPB). SPB demonstrates strong ultraviolet–visible (UV-Vis) absorptions, and is more hydrophobic than most physiological thiols. Because of these physicochemical properties it can be separated by RP-HPLC with gradient elution and detected using a diode array detector (DAD). Furthermore, we compared the derivatization reactions of H2S with MMB and MBB. Our results demonstrated the derivative effect of MMB was comparable to that of MBB. Moreover, MMB is less costly than MBB. Thus, we have developed a very sensitive method to measure the presence of H2S in plasma and tissues by derivatization using MMB. This method can provide precise quantitative information regarding the amounts of H2S associated with various normal and abnormal biochemical processes. Fig.1
2. Experimental 2.1. Reagents and solution Sodium sulfide (Na2S·9H2O) was used as a source of H2S. Stock solutions containing 1.2 mmol/L Na2S·9H2O (purchased from Sigma-Aldrich) was prepared in deionizer water (preparation from a Milli·Q Ultrapure water system) and stored at 4oC. Calibration working solutions from 0.2 to 300 nmol/mL concentration that used for quantification, recovery, precision, and accuracy studies were freshly prepared before use. The derivatization stock reagents of MMB (CAS 74235-78-2, >99%, purchased from FANBO Biochemical) and MBB (CAS 71418-44-5, ≥97%, purchased from Sigma-Aldrich) were prepared in acetonitrile at 1.51 mmol/L concentration. Tris (≥99.0%, purchased from Beijing Solarbio Science& Technology) was prepared in deionized water at 100 nmol/mL (adjust the pH by HCl to 10.1). Sulfosalicylic acid (SSA, >99.0%, purchased from Beijing chemical industry) was prepared in deionized water at 200 nmol/mL. Acetonitrile for HPLC was from Oceanpak Alexative Chemical. Formic acid was AR from Tianjin BODI chemical industry. All mobile phase solutions were filtered through 0.45 µm filters and degassed before use. 2.3 Instrumental and chromatographic conditions The chromatographic experiments were performed with an Ultimate 3000 (Thermo Scientific, USA) liquid chromatography system equipped with a vacuum membrane degasser (SRD-3600), a double ternary gradient pump (DGP-3600SDN), an auto-sampler (WPS3000SL), a column oven (TCC-3000RS), and a diode array detector (DAD-3000). The acquisition of chromatographic data was performed by means of Chromeleon software (ver.7.0, Thermo Electron). 50 µL samples were injected for each analysis. The chromatographic analysis was achieved using a C18 analytical column (Acclaim 120, 5 µm, 250×4.6 mm, Thermo Scientific, USA) with gradient elution (Table.1). Detection was performed at 390 nm. The optimized mobile phase consists of 0.1% (v/v) formic acid and acetonitrile solution. The column temperature was 30°C. Table 1 2.4 Sample preparation Blood taken from the aorta, and whole blood sample was collected, centrifuged at 3500 rpm for 15min at 4°C to obtain the plasma. Transferred the supernatant plasma to clean polypropylene tubes, and stored at −80°C until analysis. 2.5 Thiol derivatization Purging the tubes with N2 for 30 s to exhaust the air, and then 60 µL of Na2S stock or plasma were mixed with 140 µL of 0.1 mmol/L Tris-HCl (pH=10.1) solution and 60 µL of MMB (previously dissolved in acetonitrile). Immediately, the tubes were capped and the contents
were vigorously mixed for 15 s, and then incubated in the dark at 50°C. The entire procedure was carried out quickly and under the dim light. After incubation for 120 min, 60 µL SSA was added to stop the derivative reaction and precipitate protein. After centrifugation at 12,000 rpm for 12 min, the supernatants were filtered through a syringe filter (0.22 µm pore size), transferred into sample vials and analyzed using HPLC with DAD detection (λ=390 nm). 2.6 Calibration curves Calibration curves was performed using eight Na2S standards (0.2, 1, 5, 10, 50, 100, 200, and 300 nmol/mL) in the plasma. The absolute peak area was plotted against the different derivatization products concentrations, and the curves were fitted by least square linear regression analysis. 2.7 Precision and accuracy Validation of the HPLC method was performed by determining the intra-day and interday accuracy and precision. The intra-day accuracy and precision were calculated by analyzing six different runs of plasma spiked working solutions. The inter-day precision of plasma derivatization products were evaluated by the samples stored at 4 oC temperature in dark place, and determined on six replicates over three consecutive days. 2.8 Recovery For the determination of absolute recovery of derivatization products from plasma, standard solutions were prepared at three different concentrations. The samples were then treated exactly as described previously for plasma. Absolute recovery was determined by comparing the peak area obtained from the plasma to peak area obtained from the standard solutions. 2.9 Application Male Sprague-Dawley rats (180–200 g) were randomly divided into 4 groups (n=6, each) as follows: the control (Con); sodium hydrosulfide (NaHS); vitamin D3 plus nicotine (VDN); and VDN+NaHS. In these groups, VDN treatment imposed calcification, NaHS acted as an H2S donor. The rats in the VDN and VDN+NaHS groups were given vitamin D3 (300 000 IU/kg, intramuscularly) simultaneously with nicotine (25 mg/kg in 5 mL peanut oil, intragastrically) at 08:00 hours on the first day. The nicotine administration was repeated at 2000 hours. On day 2 and 15, the rats were retreated with vitamin D3. The rats of the control and VDN groups were injected with saline vehicle (0.2 mL/100 g) per day, at a volume similar to the volumes of NaHS, during the same period. The blood of all the rats were harvested on day 28, blood samples were collected in tubes, and centrifuged at 3500 rpm for 15 min at 4°C, transferred the supernatant plasma to clean polypropylene tubes, and stored at −80°C until analysis.
3. Results 3.1 Selection of derivatization conditions The derivatization conditions of MBB with Na2S solution have been reported previously [9]. MBB reacts quickly with H2S under suitable conditions. In this study, we examined various conditions for the reaction of H2S with MMB to optimize the derivatization conditions. 3.1.1. Effect of concentration of MMB The effect of the concentration of MMB on the yield of the derivatization procedure was investigated using concentrations from 0.75 to 1.5 mmol/L. The concentration and volume of Na2S used in each experiment was 200 nmol/mL and 60 µL, respectively. As is shown in Fig.2A, the maximum yield was obtained using an MMB concentration of 0.75 mmol/L. No significant differences in yield were determined in the range 0.9–1.5 mmol/L. To ensure excess of MMB, a concentration of 1.2 mmol/L was chosen to account for the concentration of HS- expected in blood samples and other reactive compounds present in biological matrices. 3.1.2. Effect of reaction time Reaction time is one of the most important factors in derivatization procedures. We tested reaction times in the range of 15–150 min (Fig.2B). Shorter reaction time especially in the range of 15-90 min can cause poor repeatability because of variability in the delays between the individual steps of the derivatization reaction. No significant changes were observed in the peak areas owing to changing reaction times from 120 to 135 min. Therefore, 120 min was selected as the reaction time for further experiments. 3.1.3. Effect of reaction temperature To determine the optimal temperature of the derivatization reaction, a wide range of temperatures was tested. The lowest temperature tested was 0°C. The highest temperature (75°C) was slightly below the boiling point of the acetonitrile reaction solvent (82°C). The reaction time was 60 min. The reaction yield increased with increasing temperature from 0 to 50°C. However, the yield decreased above 70°C. As shown in Fig. 2C, the optimal temperature was 50°C. 3.1.4. Effect of pH The pH of the reaction mixture affects the efficiency of derivatization because it determines the degree of dissociation of H2S. pKa values for the first and second dissociation steps of H2S were 7.04 and 19±2, respectively. At pH 7.4 and 25 °C about 40% of the sulfide was present as H2S. At pH 9.5, free H2S mainly exists as HS− [22]. MMB reacts with HS− at basic pH, allowing measurement of free HS−.
It has been reported previously that trace metal ion contaminations will consume sulfide in the solution or reaction vessel.[23] Nagy et al. [24]suggested lower levels of metal ion contamination in tris (hydroxymethyl) aminomethane (Tris) buffer compared to PBS ,and the linearity of sulfide could be maintained in Tris buffer . Therefore, Tris-HCl buffer has been chosen to carry out the following experiment. As shown in Fig.2D, the pH of the TrisHCl buffer was adjusted from 7.5 to 10.1 (The highest alkalinity of 100 mM Tris buffer can only reach 10.1, adding sodium hydroxide or potassium hydroxide would make the metal ion contamination higher instead.). The optimal reaction conditions were obtained to be at pH 10.1. Therefore, this pH was used in further reactions. Thiol groups are highly reactive and thus highly susceptible to oxidation by ambient oxygen. Therefore, it is therefore necessary to determine the effect of oxygen on the derivatization of H2S. A set of reaction tubes were blown with nitrogen for 0.5 min to remove air, and another set were not. Then, we conducted the derivatization procedure in each tube and determined the concentration of SPB. The results demonstrated that the reaction SPB yield was greater in the nitrogen-blown reaction tubes. Thiols were quantitatively derivatized by MMB. However, the yield was significantly reduced when the MMB reaction was conducted under visible light. Good yields were obtained when the procedure was carried out in darkness. This demonstrates that visible light has an adverse effect on the derivatization reaction with MMB. Fig. 2 3.2 HPLC separation and DAD identification The reaction product SDB has fluorescence (excitation wavelengths 390 nm, emission wavelengths 475 nm) and the maximum absorption in the near UV (λmax = 390 nm), not very different from the absorption of the reagent (λmax = 396 nm) [25]. In our study, we found that SPB similarly has strong ultraviolet–visible (UV-Vis) absorption (λmax = 390 nm) and fluorescence. When SPB was quantified by UV detector at 390 nm wavelength, the interference from other substances to the detection is minimal. Several chromatographic conditions were assessed to optimize run time, separation, and peak shape of the HPLC separation. In this study, an Acclaim 120 C18 column (200 mm×4.6 mm, 5 µm) was determined to be suitable. The composition of the eluent has a significantly effect on separation efficiency and run time. Three mobile phases were tested: acetonitrile–0.1% trifluoracetic acid (TFA), acetonitrile–0.1% trichloroacetic acid (TCA) and acetonitrile–0.1% formic acid as the mobile phase. In comparing, a mobile phase composed of 0.1% formic acid and acetonitrile containing 0.1% formic acid was found to produce optimal separation in this study. Representative chromatograms using water as the blank matrix with MMB and the standard Na2S derivative of MMB are shown in Fig. 3. These
results demonstrate that the synthesis of SPB was the result of the derivative reaction between MMB and H2S. SPB and excess MMB were completely separated within 18 min. Fig. 3 3.3 Method validation Na2S solution was reacted with MMB under the optimized experimental conditions determined previously. The calibration curve was linear from 0.2 to 300 nmol/mL using mean values (n = 3). The sensitivity of the method was satisfactory. The limit of detection was 0.03 nmol/mL at a signal-to-noise (S/N) ratio of 3/1. The limit of quantification was 0.1 nmol/mL (S/N = 10/1) (Table 2).The recoveries of the derivatized products in plasma were 94.7%~101.5% for derived products at all concentrations. Table 3 summarizes the intra- and inter-day precision and accuracy of the determinations of the derivatized products in plasma. The intra-day and inter-day precisions (%, RSD) of the measured concentrations ranged from 1.09 to 1.81% and 3.26 to 4.82%, respectively. Accuracy is often expressed in terms of Error. Error - the deviation of a measurement from its true value. The %Error observed was within 1.32%~5.26%. These results demonstrated that the sample preparation and HPLC procedure was accurate and precise. The chromatogram of SPB in plasma is shown in Fig. 4. 3.4 Comparison of the pair of isomers We compared the derivative yields of the MBB and MMB isomers. Na2S solution reacted with MMB under the optimized experimental conditions. The derivatization conditions of MBB with Na2S solution were the same as those reported previously [9]. Under the optimal chromatographic conditions in our study, as shown in Fig. 5, the SPB peak generated by derivatization with MMB was much higher than that of SDB at the same Na2S solution concentration. The LOD and correlation coefficient (R) of SPB were greater than those of SDB (Table 2). Furthermore, the interference of the impurity peak in the MMB method was less significant than that of impurity peak in the MBB method. Fig.5 3.5 Analysis of biological sample To verify the feasibility of the method, a vascular calcification (VC) rat model was established by administration of vitamin D3 plus nicotine (VDN) according to the method reported in the literature [26].Changes in H2S concentrations in the blood of different treatment groups were detected by the proposed method. Values given here represent the mean ± standard deviation of six rats in each treatment group. Statistical analyses were performed using SPSS 21.0. Comparisons between two groups were conducted using the unpaired Student’s t-test. Comparisons between ≥ 3 groups were conducted using one-way analyses of variance followed by LSD t-tests; p < 0.05 was considered statistically significant.
In the VDN group, the mean H2S content in plasma (22.48 ± 3.37 nmol/mL) was significantly lower than that of the control group (37.56 ± 4.88 nmol/mL) (p < 0.05). The H2S content in the plasma of the VDN + NaHS group (33.48 ± 2.08 nmol/mL) was greater than that of the VDN group (22.48 ± 3.37 nmol/mL) (p < 0.05). The results of the statistical analyses are illustrated as box-plots in Fig. 6. Fig. 6 4. Discussion In recent years, hydrogen sulfide, which is well known as a toxic gas, has emerged as an endogenous gaseous signaling transmitter just as nitric oxide (NO) and carbon monoxide (CO). It is shown that H2S plays a critical role in diverse physiological functions, including vascular tone, host defense against pathogens, neuromodulation, apoptosis, and energy metabolism in mammalian cells [22, 27]. The pathological and physiological significance of H2S has demonstrated the need for quantitative methods for the determination of H2S in biological media. However, the development of reliable methods for the quantitation of H2S and HS− in biological matrices has proven to be difficult because no specific physicochemical properties (strong ultraviolet-visible [UV-Vis] absorption or native fluorescence) can be exploited. In saline at physiological pH about one-third of the H2S exists as the undissociated form (H2S) and the remaining two-thirds exist as HS− in equilibrium with H2S [28]. Therefore, methods based on the selective reaction of HS− with the derivatization agent MMB have been proposed. HS− reacts with MMB by nucleophilic substitution, yielding a highly fluorescent thioether. MMB reacts quickly with H2S under suitable conditions to produce sulfide dibimane, which is more hydrophobic than most physiological thiols. Therefore, HPLC has been used for the quantitative measurement of H2S in biological media, such as plasma and tissues samples [9]. In this study, we analyzed the derivatized products by HPLC coupled with a DAD. VC is the ectopic deposition of inorganic calcium in the vascular wall. VC has been implicated in the pathogenesis of various vascular diseases and can result in devastating clinical consequences. VC is related to an increased risk of cardiovascular morbidity and related complications [29–30] and has been shown to be a strong marker of cardiovascular events in patients with diabetes and chronic kidney disease [31]. Although the mechanisms of VC are not completely understood, abnormalities in mineral metabolism are considered important risk factors. Evidence suggests that VC is a delicate and well-regulated cellular process in which vascular smooth muscle cells (VSMCs) gain an osteoblastic phenotype [32]. Extracellular phosphate (Pi) uptake through a sodium-dependent phosphate cotransporter, Pit-1, is essential for VSMC calcification and phenotypic modulation in response to elevated Pi [33]. Du et al. [34] demonstrated that H2S could dose-dependently suppress the
proliferation of VSMCs through the MAPK pathway. It has been shown that both extracellular and intracellular H2S suppresses Pi-induced calcification and osteoblastic differentiation of human aortic smooth muscle cells through suppression of Pit-1. Reduction of the endogenous production of H2S by inhibition of cystathionine γ-lyase resulted in increased osteoblastic transformation and mineralization [35]. In the present study, the H2S content of plasma of VDN rats group was lower than that of the control group. However, the decrease was less significant in VDN + NaHS-treated rats. There was no significant difference in the H2S plasma concentrations between the control group and the NaHS-treated group. The results demonstrated that our method could determine the content of H2S in biological samples sensitively and accurately. To evaluate the accuracy and applicability of the proposed method, it was successfully used to determine the concentration of H2S in tissues samples from individual rats. The vital organs (liver, kidney, spleen, lung and brain) were quickly removed, accurately weighed, and ultrasonically homogenized in an ice bath after addition of deionized water. The derivatization and chromatography conditions were carried out according to the proposed method in this paper. The precision, accuracy, and recovery of derivatized products from tissue homogenates can be found in the supplementary data (Table A.1).In addition, to further verify the accuracy of the MMB method with more representativeness, the samples (liver, kidney and plasma) have been detected and quantified by the methylene blue (MB) method and MMB method. The MB method was carried out based on the method reported in the literature [36].The MB method is probably the most commonly used method for sulfide measurement both in stock solutions and biological samples, but a weakness of the method (and all other methods that rely on colorimetric detection) is the interference of other chromophores in the sample [37-39]. It is based on the reaction of sulfide with N,Ndimethyl-p-phenylenediamine, in a ferric chloride catalyzed reaction with a 1:2 stoichiometric ratio to give the methylene blue dye, which is detected spectrophotometrically. Values given here represent the mean ± standard deviation of five rats (Table A.2). Statistical analyses were performed using SPSS 21.0. Comparisons between the groups of two methods were conducted using Wilcoxon sign-rank test. The result (p=0.074>0.05) shown that there was no statistical difference between the two methods. 5. Conclusion In summary, we have developed a simple analytical method for the analysis of H2S in biological materials. We compared the derivatization reactions of hydrogen sulfide with MMB and MBB, and developed a sensitive method to quantify H2S in blood by MMB. Selection of appropriate pH, concentration of MMB, reaction time and temperature that
would allow the method to be applied to H2S in biological materials were important factors in optimizing the derivatization procedure. Using our optimized conditions, H2S was
completely derivatized after 2 h and the derivatives were analyzed by HPLC-DAD at 390 nm. The detection levels (0.2–300 nmol/mL) may provide sufficient sensitivity for routine analysis of H2S in biological materials. To demonstrate its accuracy and applicability, the new method was successfully used to determine the residual levels of VC rats’ plasma. After evaluation of different derivatization methods (MB and MMB method), the proposed MMB method was shown that was suitable for detecting low concentrations of H2S, reliable, free of interference, precise and with a broad linear range. Acknowledgements This work was supported by The National Natural Science Foundation of China (Nos. 81573202 and Nos.81302471). References [1] J.L. Wallace, Physiological and Pathophysiological Roles of Hydrogen Sulfide in the Gastrointestinal Tract, Antioxid. Redox. Signal.12 (2010) 1125-1133. [2] J.L. Wallace, J.G.P. Ferraz, M.N. Muscara, Hydrogen sulfide: an endogenous mediator of resolution of inflammation and injury, Antioxid. Redox. Signal. 17 (2012) 58-67. [3] S.H. Cheung, W.K. Kwok, K.F. To, J.Y.W. Lau, Anti-atherogenic effect of hydrogen sulfide by over-expression of cystathionine gamma-lyase (CSE) gene, PLoS. ONE. 9 (2014) e113038. [4] S. Fiorucci, E. Distrutti, Targeting the transsulfuration-H2S pathway by FXR and GPBAR1 ligands in the treatment of portal hypertension, Pharmacol. Res. 111 (2016) 749-756. [5] X.Q. Tang, X.T. Shen, Y.E Huang, R.Q. Chen, Y.K. Ren, H.R. Fang , Y.Y. Zhuang, C.Y. Wang, Inhibition of endogenous hydrogen sulfide generation is associated with homocysteineinduced neurotoxicity: role of ERK1/2 activation, J. Mol. Neurosci. 45 (2011) 60-67. [6] M. Perry, A. Tu, G. Sehra, I. Adcock, F. Chung, The anti-proliferative effect of hydrogen sulfide upon human airway smooth muscle in COPD, Eur. Respir. J. 44 (2014) 3417 [7] S.U. Christl, H.D. Eisner, G. Dusel, H. Kasper, W. Scheppach, Antagonistic effects of sulfide and butyrate on proliferation of colonic mucosa: a potential role for these agents in the pathogenesis of ulcerative colitis, Dig. Dis. Sci. 41(1996) 2477-2481. [8] R.O. Beauchamp, J.S. Bus, J.A. Popp, C.J. Boreiko, D.A. Andjelkovich, A critical review of the literature on hydrogen sulfide toxicity, Crit. Rev. Toxicol. 13 (1984) 25–97 [9] X.G. Shen, C.B. Pattillo, S. Pardue, S.C. Bir, R. Wang, C.G. Kevil, Measurement of plasma hydrogen sulfide in vivo and in vitro, Free. Radic. Biol. Med. 50 (2011) 1021-1031.
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measure the pharmacokinetic profile of reactive sulphide species in blood, Br. J. Pharmacol. 160 (2010) 941–957. [20] X.H. Ju, S.R. Tang, Y. Jia, J.K. Guo, Y.Z. Ding, Z.G. Song, Y.J. Zhao, Determination and characterization of cysteine, glutathione and phytochelatins (PC(2)(-)(6)) in Lolium perenne L. exposed to Cd stress under ambient and elevated carbon dioxide using HPLC with fluorescence detection, J. Chromatogr. B. 879 (2011) 1717-1724. [21] X.G Shen, E.A Peter, S. Bir, R Wang, C.G. Kevil, Analytical measurement of discrete hydrogen sulfide pools in biological specimens, Free Radic. Biol. Med. 52 (2012) 2276– 2283. [22] M.N. Hughes, M. N. Centelles, K.P. Moore, Making and working with hydrogen sulfide: The chemistry and generation of hydrogen sulfide in vitro and its measurement in vivo: a review, Free Radic. Biol. Med. 47 (2009) 1346-1353. [23] I. Toth, P. Solymosi, Z. Szabo, Application of a sulfide-selective electrode in the absence of a pH-buffer, Talanta 35 (1988) 783–788. [24] P. Nagy, Z. Pálinkás, A. Nagy, B. Budai, I. Tóth, A. Vasas, Chemical aspects of hydrogen sulfide measurements in physiological samples, Biochimica et Biophysica Acta 1840 (2014) 876–891. [25] F. Sardi, B. Manta b, S.P. Ledesma, B. Knoops, M.A. Comini, G. Ferrer-Sueta, Determination of acidity and nucleophilicity in thiols by reaction with monobromobimane and fluorescence detection, Anal. Biochem. 435 (2013) 74–82. [26] R. Yang, X. Teng, X., H . Li, H.M. Xue, Q. Guo, L. Xiao, Y.M. Wu, Hydrogen Sulfide Improves Vascular Calcification in Rats by Inhibiting Endoplasmic Reticulum Stress, Oxid. Med. Cell Longev. 2016 (2016) 9095242 [27] M. Kajimura, R. Fukuda, R.M. Bateman, T. Yamamoto, M. Suematsu, Interactions of Multiple Gas-Transducing Systems: Hallmarks and Uncertainties of CO, NO, and H2S Gas Biology, Antioxid. Redox Signal. 13 (2010) 157-192. [28] R. Hosoki, N. Matsuki, H. Kimura, The Possible Role of Hydrogen Sulfide as an Endogenous Smooth Muscle Relaxant in Synergy with Nitric Oxide, Biochem. Biophys. Res. Commun. 237 (1997) 527-531. [29] J.A. Rumberger, D.B. Simons, L.A. Fitzpatrick, P.F. Sheedy, R.S. Schwartz, Coronary artery calcium area by electron-beam computed tomography and coronary atherosclerotic plaque area: A histopathologic correlative study, Circulation. 92 (1995) 2157-2162.
[30] P.J. Fitzgerald, Ports T.A., P.G. Yock , Contribution of localized calcium deposits to dissection after angioplasty. An observational study using intravascular ultrasound, Circulation 86 (1992) 64-70. [31] S. Lehto, L. Niskanen, M. Suhonen, T. Ronnemaa, M. Laakso, Medial artery calcification: a neglected harbinger of cardiovascular complications in non–insulindependent diabetes mellitus, Arterioscler Thromb. Vasc. Biol. 16 (1996) 978-983. [32] K.A. Lomashvili, S. Cobbs, R.A. Hennigar, K.I. Hardcastle, W.C.O'Neill, Phosphate-Induced Vascular Calcification: Role of Pyrophosphate and Osteopontin, J. Am. Soc. Nephrol. 15 (2004) 1392-1401. [33] X.Li, H.Y.Yang, C.M. Giachelli, Role of the sodium-dependent phosphate cotransporter, Pit-1, in vascular smooth muscle cell calcification, Circ. Res. 98(2006) 905-912. [34] J.B. Du, Y. Hui, Y.F. Cheung, G. Bin, H.F. Jiang, X.B. Chen, C.S. Tang, The possible role of hydrogen sulfide as a smooth muscle cell proliferation inhibitor in rat cultured cells, Heart Vessels. 19 (2004) 75-80. [35] E. Zavaczki, V. Jeney, A. Agarwal, A. Zarjou, M. Oros, M. Katko´, Z. Varga, G. Balla, J. Balla, Hydrogen sulfide inhibits the calcification and osteoblastic differentiation of vascular smooth muscle cells, Kidney Int. 80 (2011) 731-739. [36] Y.Y.P Mok, M.S.B.M Atan, C.Y Ping, W.Z Jing, M Bhatia, S Moochhala, P.K. Moore, Role of hydrogen sulphide in haemorrhagic shock in the rat: protective effect of inhibitors of hydrogen sulphide biosynthesis, Br. J. Pharmacol. 143 (2004) 881–889. [37] M.H. Stipanuk, P.W. Beck, Characterization of the enzymic capacity for cysteine desulphhydration in liver and kidney of the rat, Biochem. J. 206 (1982) 267. [38] S. Rashid, J.K. Heer, M.J. Garle, S.P.H Alexander, R.E. Roberts, Hydrogen sulphideinduced relaxation of porcine peripheral bronchioles, Br. J. Pharmacol. 168 (2013) 1902–1910. [39] Roberta d'Emmanuele di Villa Bianca, Emma Mitidieri , E. Donnarumma , T. Tramontano , V. Brancaleone , G. Cirino, M. Bucci , R. Sorrentino. Hydrogen sulfide is involved in dexamethasone-induced hypertension in rat, Nitric Oxide. 46 (2015) 80–86.
Table1 The gradient elution program Time(min)
Rates(mL/min)
A (0.1% formic acid, %)
B (acetonitrile, %)
0
0.6
85
15
3
0.6
68
32
16
0.6
55
45
17
0.6
85
15
18
0.6
85
15
Table 2 Equations of calibration curves and LOD used for the analysis of MBB and MMB method
a
Derivation
Derivation
Concentrations
Agent
Products
(nmol/mL)
LOD Equationa
Correlation (R)
(nmol/mL, S/N=3)
MBB
SDB
1~200
Y=0.0289X+0.6428
0.9914
0.4
MMB
SPB
0.2~300
Y=0.0488X-0.2060
0.9991
0.03
X and Y are the concentration of Na2S and peak area, respectively.
Table 3 The precision, accuracy and recovery of derivatized products in plasma. Actual Sample
plasma
Added
Calculate
Found b
Recovery
Accuracy
c
d
L)
(%)
(%)
da (nmol/m
(nmol/m
L)
L)
36.478
(nmol/m L)
(nmol/m
Precisione(%)
Intra-day
Inter-day
2.5
20.739
19.647
94.7
5.26
1.09
3.62
50
68.239
69.290
101.5
1.54
1.65
3.26
150
168.239
166.022
98.7
1.32
1.81
4.82
a. The calculation concentration of plasma samples was calculated by 30 µL plasma spiked 30 µL three concentrations of working solutions. b. Results are mean of three runs. c. Recovery (%) = (found concentration obtained from sample spiked with Na2S)/(calculation
concentration
obtained
from
sample
spiked
standard
solution)×100% d. Accuracy is expressed in terms of error. ERROR (%) = (found concentrationcalculated concentration)/ (calculated concentration) ×100%. e. Relative standard deviation (R.S.D.). The derivative plasma samples were measured for the inter-day accuracy and the intraday precision within 3 days with three repetitions each.
Figures captions: Figure 1 Structures of monobromobimane isomers. Figure 2 Effects of (A) concentration of MMB, (B) time, (C) temperature, and (D) pH on the derivatization of hydrogen sulfide by MMB. Figure 3 Typical chromatograms of water with (A) MMB and (B) standard Na2S derivative of MMB. Figure 4 Chromatogram of SPB in plasma. Figure 5 The proposed reaction of HS−, and chromatograms of (A) SPB and (B) SDB generated by derivatization reactions with 100 nmol/mL Na2S solution. Figure 6 Changes in H2S concentration in plasma of different rat groups. Values represent mean ± S.D. (n = 6). VDN group vs. control group; vs. calcification group, p < 0.05. NaHS group vs. control group vs. VDN + NaHS group, p > 0.05.
Br
Br
O
O
N N
N N
O
O
MMB
MBB
Fig.1
0.3
0.22
B
A
Peak Area
Peak Area
0.20
0.18
0.16
0.8
1.0
1.2
1.4
1.6
0.2
0.1
0.0 0
30
MMB Concentration(mmol/L)
60
90
120
150
Time(min) 0.4
0.6 C
D
0.3
Paek Area
Peak Area
0.4 0.2
0.2 0.1 0.0
0.0 0
20
40
60
Temperature(
℃
)
Fig.2
80
7.5
8.0
8.5
9.0 pH
9.5
10.0
10.5
10.00
A
mAU
7.50 5.00 2.50 0.00
min 0.0
10.00
5.0
10.0
15.0
mAU
18.0
B
7.50 5.00 SPB 2.50 0.0
min 0.0
5.0
10.0
Fig.3
15.0
18.0
15.0 mAU
0.0 0.0
SPB
5.0
10.0
Fig.4
.
15.0
min
B
A
Br Br
O
O
+ HS
N N
+ HS-
N N
-
O
O
pH =9.5 pH =10.1
O
O N N
S
O
N N
N N
S
O N N
O
O
O O
6.00
6.00
mAU
4.00
4.00 2.00
mAU
SPB
2.00 SDB
0.00 10.00
15.00
0.00 min 18.00 10.00
Figure 5
min
15.00
18.00
Fig.6
S1570-0232(16)31327-7 http://dx.doi.org/doi:10.1016/j.jchromb.2017.04.023 CHROMB 20562
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
26-11-2016 28-2-2017 12-4-2017
Please cite this article as: Xiao-Xin Tan, Kao-Qi Lian, Xiang Li, Nan Li, Wei Wang, Wei-Jun Kang, Hong-Mei Shi, Development of a derivatization method for the quantification of hydrogen sulfide and its application in vascular calcification rats, Journal of Chromatography Bhttp://dx.doi.org/10.1016/j.jchromb.2017.04.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development of a derivatization method for the quantification of hydrogen sulfide and its application in vascular calcification rats Xiao-Xin Tan 1, Kao-Qi Lian 1, Xiang Li, Nan Li, Wei Wang, Wei-Jun Kang, Hong-Mei Shi * Department of Health inspection, School of Public Health, Hebei Medical University, Shijiazhuang 050017, China
Highlights:
Areliable and sensitive HPLC method for the determination of H2S was reported.
The method is sensitive enough to be applied in plasma and various tissues.
The derivatization reaction for H2S by the isomers (MMB and MBB) was first compared.
The proposed method has been applicable for studies in the VC model of rats.
It might be an useful tool for the investigation on the pathological of H2S.
Abstract: Hydrogen sulfide (H2S) plays major functional and structural roles in diverse physiological functions and the pathogenesis of a variety of disorders in biological matrices. The significance of H2S has prompted the development of sensitive and selective methods to determine its concentration in biological samples. The fluorescent reagent monobromobimane (MBB) has been widely used to measure various thiol-containing species through alkylation. MBB may prevent the oxidation of sulfide and the reaction of sulfide with several different species (such as superoxide radicals, hydrogen peroxide and peroxynitrite). An isomers of MBB, 3-(bromomethyl)-2, 6, 7-trimethyl-1H, 5H-pyrazolo [1, 2a] pyrazole-1, 5-dione (MMB), is cheaper than MBB and its use in the analysis of H2S has not
1
These authors contributed equally to this work.
*Corresponding author. Addresses: Department of sanitary inspection, School of Public
Health, Hebei Medical University, East Zhongshan Road 361, Shijiazhuang, P. R.China. E-mail: [email protected]. (H.M. Shi).
previously been reported. In the present study, we compared the derivatization reactions of hydrogen sulfide with MMB and MBB and developed a sensitive method to quantify H2S in blood. In our method, H2S was incubated in the dark with excess MMB in 0.1M Tris-HCl buffer (pH 10.1) at 50 ℃ for 120min. 50 µL aliquots of the derivatized product were analyzed using HPLC system with gradient elution of 0.1% (v/v) formic acid-acetonitrile. The limit of detection for the derivatized product was 0.03 nmol/mL. The derivatization reaction was suitable for detecting low concentrations of H2S. The derivate product is stable over time, permitting batch storage and analysis. Keywords：Hydrogen sulfide; Monobromobimane; Isomers; Derivatization; HPLC; Vascular calcification 1. Introduction
Hydrogen sulfide (H2S) is produced from a variety of sources. It is a colorless, flammable, water-soluble gas with an offensive odor. H2S is produced during amino acid metabolism by the trans-sulfuration and cysteine desulfuration pathways. H2S production is predominantly catalyzed by three tissue-specific enzymes, cystathionine β-synthase
(CBS),
cystathionine
γ-lyase
(CSE),
and
3-mercaptopyruvate
sulfurtransferase (3MST). In recent years, H2S has received increased research attention along with other gaseous mediators (such as nitric oxide and carbon monoxide), as it plays a critical role in diverse physiological functions, including neuronal, inflammatory processes, cardiovascular and endocrine processes [1-3]. Hydrogen sulfide has also been reported to participate in the pathogenesis of a variety of disorders, including portal hypertension, Alzheimer’s disease, pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD) and ulcerative colitis [4-7]. Accurately and reliably measuring the concentration of biologically free H2S in biological matrices can
provide information regarding the amounts involved in physiological and pathological responses. H2S can exist as different species, depending on the pH of the surrounding environment. In aqueous solution, free H2S is a weak acid with two acid dissociation constants [8]. In vivo, H2S exists primarily as the highly reactive hydrosulfide (HS−) anion because of the mildly basic pH. Furthermore, the oxidation of sulfide and its reaction with species such as superoxide radicals, hydrogen peroxide, and peroxynitrite, adversely affect the determination of absolute H2S in biological samples [9].It is rare to have a consensus for the concentrations of H2S associated with normal or pathological physiological processes. The unstable nature of H2S in solution makes measurement and analysis of H2S in biological matrices difficult. Several different methods have been developed for detecting H2S, including electrochemical and colorimetric methods [10-13], gas chromatography [14-15] and derivatization methods[16], using pentafluorobenzyl bromide [17] or HF-PBA [18], for example. Because of the susceptibility of sulfide to oxidation, and the reversible nature of sulfide bonds in blood and tissues, we sought to develop a simple method that quickly captures reactive sulfide without releasing chemically bound sulfur within a biological matrix, to obtain measurements that accurately quantify biologically available sulfide. The fluorescent reagent monobromobimane (MBB) has been widely used to measure various thiol-containing species through alkylation [19-20]. MBB can reacts with HS− under suitable conditions to produce sulfide-dibimane (SDB). SDB allows for the quick and accurate determination of absolute H2S present in the biological media [9, 19, and 21]. 3(bromomethyl)-2, 6, 7-trimethyl-1H, 5H-pyrazolo [1, 2-a] pyrazole-1, 5-dione (MMB) is an isomer of MBB (the structural formulas are shown in Fig.1). In this study, we found that MMB reacted readily with HS− under suitable conditions to produce sulfide-pardimane (SPB). SPB demonstrates strong ultraviolet–visible (UV-Vis) absorptions, and is more hydrophobic than most physiological thiols. Because of these physicochemical properties it can be separated by RP-HPLC with gradient elution and detected using a diode array detector (DAD). Furthermore, we compared the derivatization reactions of H2S with MMB and MBB. Our results demonstrated the derivative effect of MMB was comparable to that of MBB. Moreover, MMB is less costly than MBB. Thus, we have developed a very sensitive method to measure the presence of H2S in plasma and tissues by derivatization using MMB. This method can provide precise quantitative information regarding the amounts of H2S associated with various normal and abnormal biochemical processes. Fig.1
2. Experimental 2.1. Reagents and solution Sodium sulfide (Na2S·9H2O) was used as a source of H2S. Stock solutions containing 1.2 mmol/L Na2S·9H2O (purchased from Sigma-Aldrich) was prepared in deionizer water (preparation from a Milli·Q Ultrapure water system) and stored at 4oC. Calibration working solutions from 0.2 to 300 nmol/mL concentration that used for quantification, recovery, precision, and accuracy studies were freshly prepared before use. The derivatization stock reagents of MMB (CAS 74235-78-2, >99%, purchased from FANBO Biochemical) and MBB (CAS 71418-44-5, ≥97%, purchased from Sigma-Aldrich) were prepared in acetonitrile at 1.51 mmol/L concentration. Tris (≥99.0%, purchased from Beijing Solarbio Science& Technology) was prepared in deionized water at 100 nmol/mL (adjust the pH by HCl to 10.1). Sulfosalicylic acid (SSA, >99.0%, purchased from Beijing chemical industry) was prepared in deionized water at 200 nmol/mL. Acetonitrile for HPLC was from Oceanpak Alexative Chemical. Formic acid was AR from Tianjin BODI chemical industry. All mobile phase solutions were filtered through 0.45 µm filters and degassed before use. 2.3 Instrumental and chromatographic conditions The chromatographic experiments were performed with an Ultimate 3000 (Thermo Scientific, USA) liquid chromatography system equipped with a vacuum membrane degasser (SRD-3600), a double ternary gradient pump (DGP-3600SDN), an auto-sampler (WPS3000SL), a column oven (TCC-3000RS), and a diode array detector (DAD-3000). The acquisition of chromatographic data was performed by means of Chromeleon software (ver.7.0, Thermo Electron). 50 µL samples were injected for each analysis. The chromatographic analysis was achieved using a C18 analytical column (Acclaim 120, 5 µm, 250×4.6 mm, Thermo Scientific, USA) with gradient elution (Table.1). Detection was performed at 390 nm. The optimized mobile phase consists of 0.1% (v/v) formic acid and acetonitrile solution. The column temperature was 30°C. Table 1 2.4 Sample preparation Blood taken from the aorta, and whole blood sample was collected, centrifuged at 3500 rpm for 15min at 4°C to obtain the plasma. Transferred the supernatant plasma to clean polypropylene tubes, and stored at −80°C until analysis. 2.5 Thiol derivatization Purging the tubes with N2 for 30 s to exhaust the air, and then 60 µL of Na2S stock or plasma were mixed with 140 µL of 0.1 mmol/L Tris-HCl (pH=10.1) solution and 60 µL of MMB (previously dissolved in acetonitrile). Immediately, the tubes were capped and the contents
were vigorously mixed for 15 s, and then incubated in the dark at 50°C. The entire procedure was carried out quickly and under the dim light. After incubation for 120 min, 60 µL SSA was added to stop the derivative reaction and precipitate protein. After centrifugation at 12,000 rpm for 12 min, the supernatants were filtered through a syringe filter (0.22 µm pore size), transferred into sample vials and analyzed using HPLC with DAD detection (λ=390 nm). 2.6 Calibration curves Calibration curves was performed using eight Na2S standards (0.2, 1, 5, 10, 50, 100, 200, and 300 nmol/mL) in the plasma. The absolute peak area was plotted against the different derivatization products concentrations, and the curves were fitted by least square linear regression analysis. 2.7 Precision and accuracy Validation of the HPLC method was performed by determining the intra-day and interday accuracy and precision. The intra-day accuracy and precision were calculated by analyzing six different runs of plasma spiked working solutions. The inter-day precision of plasma derivatization products were evaluated by the samples stored at 4 oC temperature in dark place, and determined on six replicates over three consecutive days. 2.8 Recovery For the determination of absolute recovery of derivatization products from plasma, standard solutions were prepared at three different concentrations. The samples were then treated exactly as described previously for plasma. Absolute recovery was determined by comparing the peak area obtained from the plasma to peak area obtained from the standard solutions. 2.9 Application Male Sprague-Dawley rats (180–200 g) were randomly divided into 4 groups (n=6, each) as follows: the control (Con); sodium hydrosulfide (NaHS); vitamin D3 plus nicotine (VDN); and VDN+NaHS. In these groups, VDN treatment imposed calcification, NaHS acted as an H2S donor. The rats in the VDN and VDN+NaHS groups were given vitamin D3 (300 000 IU/kg, intramuscularly) simultaneously with nicotine (25 mg/kg in 5 mL peanut oil, intragastrically) at 08:00 hours on the first day. The nicotine administration was repeated at 2000 hours. On day 2 and 15, the rats were retreated with vitamin D3. The rats of the control and VDN groups were injected with saline vehicle (0.2 mL/100 g) per day, at a volume similar to the volumes of NaHS, during the same period. The blood of all the rats were harvested on day 28, blood samples were collected in tubes, and centrifuged at 3500 rpm for 15 min at 4°C, transferred the supernatant plasma to clean polypropylene tubes, and stored at −80°C until analysis.
3. Results 3.1 Selection of derivatization conditions The derivatization conditions of MBB with Na2S solution have been reported previously [9]. MBB reacts quickly with H2S under suitable conditions. In this study, we examined various conditions for the reaction of H2S with MMB to optimize the derivatization conditions. 3.1.1. Effect of concentration of MMB The effect of the concentration of MMB on the yield of the derivatization procedure was investigated using concentrations from 0.75 to 1.5 mmol/L. The concentration and volume of Na2S used in each experiment was 200 nmol/mL and 60 µL, respectively. As is shown in Fig.2A, the maximum yield was obtained using an MMB concentration of 0.75 mmol/L. No significant differences in yield were determined in the range 0.9–1.5 mmol/L. To ensure excess of MMB, a concentration of 1.2 mmol/L was chosen to account for the concentration of HS- expected in blood samples and other reactive compounds present in biological matrices. 3.1.2. Effect of reaction time Reaction time is one of the most important factors in derivatization procedures. We tested reaction times in the range of 15–150 min (Fig.2B). Shorter reaction time especially in the range of 15-90 min can cause poor repeatability because of variability in the delays between the individual steps of the derivatization reaction. No significant changes were observed in the peak areas owing to changing reaction times from 120 to 135 min. Therefore, 120 min was selected as the reaction time for further experiments. 3.1.3. Effect of reaction temperature To determine the optimal temperature of the derivatization reaction, a wide range of temperatures was tested. The lowest temperature tested was 0°C. The highest temperature (75°C) was slightly below the boiling point of the acetonitrile reaction solvent (82°C). The reaction time was 60 min. The reaction yield increased with increasing temperature from 0 to 50°C. However, the yield decreased above 70°C. As shown in Fig. 2C, the optimal temperature was 50°C. 3.1.4. Effect of pH The pH of the reaction mixture affects the efficiency of derivatization because it determines the degree of dissociation of H2S. pKa values for the first and second dissociation steps of H2S were 7.04 and 19±2, respectively. At pH 7.4 and 25 °C about 40% of the sulfide was present as H2S. At pH 9.5, free H2S mainly exists as HS− [22]. MMB reacts with HS− at basic pH, allowing measurement of free HS−.
It has been reported previously that trace metal ion contaminations will consume sulfide in the solution or reaction vessel.[23] Nagy et al. [24]suggested lower levels of metal ion contamination in tris (hydroxymethyl) aminomethane (Tris) buffer compared to PBS ,and the linearity of sulfide could be maintained in Tris buffer . Therefore, Tris-HCl buffer has been chosen to carry out the following experiment. As shown in Fig.2D, the pH of the TrisHCl buffer was adjusted from 7.5 to 10.1 (The highest alkalinity of 100 mM Tris buffer can only reach 10.1, adding sodium hydroxide or potassium hydroxide would make the metal ion contamination higher instead.). The optimal reaction conditions were obtained to be at pH 10.1. Therefore, this pH was used in further reactions. Thiol groups are highly reactive and thus highly susceptible to oxidation by ambient oxygen. Therefore, it is therefore necessary to determine the effect of oxygen on the derivatization of H2S. A set of reaction tubes were blown with nitrogen for 0.5 min to remove air, and another set were not. Then, we conducted the derivatization procedure in each tube and determined the concentration of SPB. The results demonstrated that the reaction SPB yield was greater in the nitrogen-blown reaction tubes. Thiols were quantitatively derivatized by MMB. However, the yield was significantly reduced when the MMB reaction was conducted under visible light. Good yields were obtained when the procedure was carried out in darkness. This demonstrates that visible light has an adverse effect on the derivatization reaction with MMB. Fig. 2 3.2 HPLC separation and DAD identification The reaction product SDB has fluorescence (excitation wavelengths 390 nm, emission wavelengths 475 nm) and the maximum absorption in the near UV (λmax = 390 nm), not very different from the absorption of the reagent (λmax = 396 nm) [25]. In our study, we found that SPB similarly has strong ultraviolet–visible (UV-Vis) absorption (λmax = 390 nm) and fluorescence. When SPB was quantified by UV detector at 390 nm wavelength, the interference from other substances to the detection is minimal. Several chromatographic conditions were assessed to optimize run time, separation, and peak shape of the HPLC separation. In this study, an Acclaim 120 C18 column (200 mm×4.6 mm, 5 µm) was determined to be suitable. The composition of the eluent has a significantly effect on separation efficiency and run time. Three mobile phases were tested: acetonitrile–0.1% trifluoracetic acid (TFA), acetonitrile–0.1% trichloroacetic acid (TCA) and acetonitrile–0.1% formic acid as the mobile phase. In comparing, a mobile phase composed of 0.1% formic acid and acetonitrile containing 0.1% formic acid was found to produce optimal separation in this study. Representative chromatograms using water as the blank matrix with MMB and the standard Na2S derivative of MMB are shown in Fig. 3. These
results demonstrate that the synthesis of SPB was the result of the derivative reaction between MMB and H2S. SPB and excess MMB were completely separated within 18 min. Fig. 3 3.3 Method validation Na2S solution was reacted with MMB under the optimized experimental conditions determined previously. The calibration curve was linear from 0.2 to 300 nmol/mL using mean values (n = 3). The sensitivity of the method was satisfactory. The limit of detection was 0.03 nmol/mL at a signal-to-noise (S/N) ratio of 3/1. The limit of quantification was 0.1 nmol/mL (S/N = 10/1) (Table 2).The recoveries of the derivatized products in plasma were 94.7%~101.5% for derived products at all concentrations. Table 3 summarizes the intra- and inter-day precision and accuracy of the determinations of the derivatized products in plasma. The intra-day and inter-day precisions (%, RSD) of the measured concentrations ranged from 1.09 to 1.81% and 3.26 to 4.82%, respectively. Accuracy is often expressed in terms of Error. Error - the deviation of a measurement from its true value. The %Error observed was within 1.32%~5.26%. These results demonstrated that the sample preparation and HPLC procedure was accurate and precise. The chromatogram of SPB in plasma is shown in Fig. 4. 3.4 Comparison of the pair of isomers We compared the derivative yields of the MBB and MMB isomers. Na2S solution reacted with MMB under the optimized experimental conditions. The derivatization conditions of MBB with Na2S solution were the same as those reported previously [9]. Under the optimal chromatographic conditions in our study, as shown in Fig. 5, the SPB peak generated by derivatization with MMB was much higher than that of SDB at the same Na2S solution concentration. The LOD and correlation coefficient (R) of SPB were greater than those of SDB (Table 2). Furthermore, the interference of the impurity peak in the MMB method was less significant than that of impurity peak in the MBB method. Fig.5 3.5 Analysis of biological sample To verify the feasibility of the method, a vascular calcification (VC) rat model was established by administration of vitamin D3 plus nicotine (VDN) according to the method reported in the literature [26].Changes in H2S concentrations in the blood of different treatment groups were detected by the proposed method. Values given here represent the mean ± standard deviation of six rats in each treatment group. Statistical analyses were performed using SPSS 21.0. Comparisons between two groups were conducted using the unpaired Student’s t-test. Comparisons between ≥ 3 groups were conducted using one-way analyses of variance followed by LSD t-tests; p < 0.05 was considered statistically significant.
In the VDN group, the mean H2S content in plasma (22.48 ± 3.37 nmol/mL) was significantly lower than that of the control group (37.56 ± 4.88 nmol/mL) (p < 0.05). The H2S content in the plasma of the VDN + NaHS group (33.48 ± 2.08 nmol/mL) was greater than that of the VDN group (22.48 ± 3.37 nmol/mL) (p < 0.05). The results of the statistical analyses are illustrated as box-plots in Fig. 6. Fig. 6 4. Discussion In recent years, hydrogen sulfide, which is well known as a toxic gas, has emerged as an endogenous gaseous signaling transmitter just as nitric oxide (NO) and carbon monoxide (CO). It is shown that H2S plays a critical role in diverse physiological functions, including vascular tone, host defense against pathogens, neuromodulation, apoptosis, and energy metabolism in mammalian cells [22, 27]. The pathological and physiological significance of H2S has demonstrated the need for quantitative methods for the determination of H2S in biological media. However, the development of reliable methods for the quantitation of H2S and HS− in biological matrices has proven to be difficult because no specific physicochemical properties (strong ultraviolet-visible [UV-Vis] absorption or native fluorescence) can be exploited. In saline at physiological pH about one-third of the H2S exists as the undissociated form (H2S) and the remaining two-thirds exist as HS− in equilibrium with H2S [28]. Therefore, methods based on the selective reaction of HS− with the derivatization agent MMB have been proposed. HS− reacts with MMB by nucleophilic substitution, yielding a highly fluorescent thioether. MMB reacts quickly with H2S under suitable conditions to produce sulfide dibimane, which is more hydrophobic than most physiological thiols. Therefore, HPLC has been used for the quantitative measurement of H2S in biological media, such as plasma and tissues samples [9]. In this study, we analyzed the derivatized products by HPLC coupled with a DAD. VC is the ectopic deposition of inorganic calcium in the vascular wall. VC has been implicated in the pathogenesis of various vascular diseases and can result in devastating clinical consequences. VC is related to an increased risk of cardiovascular morbidity and related complications [29–30] and has been shown to be a strong marker of cardiovascular events in patients with diabetes and chronic kidney disease [31]. Although the mechanisms of VC are not completely understood, abnormalities in mineral metabolism are considered important risk factors. Evidence suggests that VC is a delicate and well-regulated cellular process in which vascular smooth muscle cells (VSMCs) gain an osteoblastic phenotype [32]. Extracellular phosphate (Pi) uptake through a sodium-dependent phosphate cotransporter, Pit-1, is essential for VSMC calcification and phenotypic modulation in response to elevated Pi [33]. Du et al. [34] demonstrated that H2S could dose-dependently suppress the
proliferation of VSMCs through the MAPK pathway. It has been shown that both extracellular and intracellular H2S suppresses Pi-induced calcification and osteoblastic differentiation of human aortic smooth muscle cells through suppression of Pit-1. Reduction of the endogenous production of H2S by inhibition of cystathionine γ-lyase resulted in increased osteoblastic transformation and mineralization [35]. In the present study, the H2S content of plasma of VDN rats group was lower than that of the control group. However, the decrease was less significant in VDN + NaHS-treated rats. There was no significant difference in the H2S plasma concentrations between the control group and the NaHS-treated group. The results demonstrated that our method could determine the content of H2S in biological samples sensitively and accurately. To evaluate the accuracy and applicability of the proposed method, it was successfully used to determine the concentration of H2S in tissues samples from individual rats. The vital organs (liver, kidney, spleen, lung and brain) were quickly removed, accurately weighed, and ultrasonically homogenized in an ice bath after addition of deionized water. The derivatization and chromatography conditions were carried out according to the proposed method in this paper. The precision, accuracy, and recovery of derivatized products from tissue homogenates can be found in the supplementary data (Table A.1).In addition, to further verify the accuracy of the MMB method with more representativeness, the samples (liver, kidney and plasma) have been detected and quantified by the methylene blue (MB) method and MMB method. The MB method was carried out based on the method reported in the literature [36].The MB method is probably the most commonly used method for sulfide measurement both in stock solutions and biological samples, but a weakness of the method (and all other methods that rely on colorimetric detection) is the interference of other chromophores in the sample [37-39]. It is based on the reaction of sulfide with N,Ndimethyl-p-phenylenediamine, in a ferric chloride catalyzed reaction with a 1:2 stoichiometric ratio to give the methylene blue dye, which is detected spectrophotometrically. Values given here represent the mean ± standard deviation of five rats (Table A.2). Statistical analyses were performed using SPSS 21.0. Comparisons between the groups of two methods were conducted using Wilcoxon sign-rank test. The result (p=0.074>0.05) shown that there was no statistical difference between the two methods. 5. Conclusion In summary, we have developed a simple analytical method for the analysis of H2S in biological materials. We compared the derivatization reactions of hydrogen sulfide with MMB and MBB, and developed a sensitive method to quantify H2S in blood by MMB. Selection of appropriate pH, concentration of MMB, reaction time and temperature that
would allow the method to be applied to H2S in biological materials were important factors in optimizing the derivatization procedure. Using our optimized conditions, H2S was
completely derivatized after 2 h and the derivatives were analyzed by HPLC-DAD at 390 nm. The detection levels (0.2–300 nmol/mL) may provide sufficient sensitivity for routine analysis of H2S in biological materials. To demonstrate its accuracy and applicability, the new method was successfully used to determine the residual levels of VC rats’ plasma. After evaluation of different derivatization methods (MB and MMB method), the proposed MMB method was shown that was suitable for detecting low concentrations of H2S, reliable, free of interference, precise and with a broad linear range. Acknowledgements This work was supported by The National Natural Science Foundation of China (Nos. 81573202 and Nos.81302471). References [1] J.L. Wallace, Physiological and Pathophysiological Roles of Hydrogen Sulfide in the Gastrointestinal Tract, Antioxid. Redox. Signal.12 (2010) 1125-1133. [2] J.L. Wallace, J.G.P. Ferraz, M.N. Muscara, Hydrogen sulfide: an endogenous mediator of resolution of inflammation and injury, Antioxid. Redox. Signal. 17 (2012) 58-67. [3] S.H. Cheung, W.K. Kwok, K.F. To, J.Y.W. Lau, Anti-atherogenic effect of hydrogen sulfide by over-expression of cystathionine gamma-lyase (CSE) gene, PLoS. ONE. 9 (2014) e113038. [4] S. Fiorucci, E. Distrutti, Targeting the transsulfuration-H2S pathway by FXR and GPBAR1 ligands in the treatment of portal hypertension, Pharmacol. Res. 111 (2016) 749-756. [5] X.Q. Tang, X.T. Shen, Y.E Huang, R.Q. Chen, Y.K. Ren, H.R. Fang , Y.Y. Zhuang, C.Y. Wang, Inhibition of endogenous hydrogen sulfide generation is associated with homocysteineinduced neurotoxicity: role of ERK1/2 activation, J. Mol. Neurosci. 45 (2011) 60-67. [6] M. Perry, A. Tu, G. Sehra, I. Adcock, F. Chung, The anti-proliferative effect of hydrogen sulfide upon human airway smooth muscle in COPD, Eur. Respir. J. 44 (2014) 3417 [7] S.U. Christl, H.D. Eisner, G. Dusel, H. Kasper, W. Scheppach, Antagonistic effects of sulfide and butyrate on proliferation of colonic mucosa: a potential role for these agents in the pathogenesis of ulcerative colitis, Dig. Dis. Sci. 41(1996) 2477-2481. [8] R.O. Beauchamp, J.S. Bus, J.A. Popp, C.J. Boreiko, D.A. Andjelkovich, A critical review of the literature on hydrogen sulfide toxicity, Crit. Rev. Toxicol. 13 (1984) 25–97 [9] X.G. Shen, C.B. Pattillo, S. Pardue, S.C. Bir, R. Wang, C.G. Kevil, Measurement of plasma hydrogen sulfide in vivo and in vitro, Free. Radic. Biol. Med. 50 (2011) 1021-1031.
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Table1 The gradient elution program Time(min)
Rates(mL/min)
A (0.1% formic acid, %)
B (acetonitrile, %)
0
0.6
85
15
3
0.6
68
32
16
0.6
55
45
17
0.6
85
15
18
0.6
85
15
Table 2 Equations of calibration curves and LOD used for the analysis of MBB and MMB method
a
Derivation
Derivation
Concentrations
Agent
Products
(nmol/mL)
LOD Equationa
Correlation (R)
(nmol/mL, S/N=3)
MBB
SDB
1~200
Y=0.0289X+0.6428
0.9914
0.4
MMB
SPB
0.2~300
Y=0.0488X-0.2060
0.9991
0.03
X and Y are the concentration of Na2S and peak area, respectively.
Table 3 The precision, accuracy and recovery of derivatized products in plasma. Actual Sample
plasma
Added
Calculate
Found b
Recovery
Accuracy
c
d
L)
(%)
(%)
da (nmol/m
(nmol/m
L)
L)
36.478
(nmol/m L)
(nmol/m
Precisione(%)
Intra-day
Inter-day
2.5
20.739
19.647
94.7
5.26
1.09
3.62
50
68.239
69.290
101.5
1.54
1.65
3.26
150
168.239
166.022
98.7
1.32
1.81
4.82
a. The calculation concentration of plasma samples was calculated by 30 µL plasma spiked 30 µL three concentrations of working solutions. b. Results are mean of three runs. c. Recovery (%) = (found concentration obtained from sample spiked with Na2S)/(calculation
concentration
obtained
from
sample
spiked
standard
solution)×100% d. Accuracy is expressed in terms of error. ERROR (%) = (found concentrationcalculated concentration)/ (calculated concentration) ×100%. e. Relative standard deviation (R.S.D.). The derivative plasma samples were measured for the inter-day accuracy and the intraday precision within 3 days with three repetitions each.
Figures captions: Figure 1 Structures of monobromobimane isomers. Figure 2 Effects of (A) concentration of MMB, (B) time, (C) temperature, and (D) pH on the derivatization of hydrogen sulfide by MMB. Figure 3 Typical chromatograms of water with (A) MMB and (B) standard Na2S derivative of MMB. Figure 4 Chromatogram of SPB in plasma. Figure 5 The proposed reaction of HS−, and chromatograms of (A) SPB and (B) SDB generated by derivatization reactions with 100 nmol/mL Na2S solution. Figure 6 Changes in H2S concentration in plasma of different rat groups. Values represent mean ± S.D. (n = 6). VDN group vs. control group; vs. calcification group, p < 0.05. NaHS group vs. control group vs. VDN + NaHS group, p > 0.05.
Br
Br
O
O
N N
N N
O
O
MMB
MBB
Fig.1
0.3
0.22
B
A
Peak Area
Peak Area
0.20
0.18
0.16
0.8
1.0
1.2
1.4
1.6
0.2
0.1
0.0 0
30
MMB Concentration(mmol/L)
60
90
120
150
Time(min) 0.4
0.6 C
D
0.3
Paek Area
Peak Area
0.4 0.2
0.2 0.1 0.0
0.0 0
20
40
60
Temperature(
℃
)
Fig.2
80
7.5
8.0
8.5
9.0 pH
9.5
10.0
10.5
10.00
A
mAU
7.50 5.00 2.50 0.00
min 0.0
10.00
5.0
10.0
15.0
mAU
18.0
B
7.50 5.00 SPB 2.50 0.0
min 0.0
5.0
10.0
Fig.3
15.0
18.0
15.0 mAU
0.0 0.0
SPB
5.0
10.0
Fig.4
.
15.0
min
B
A
Br Br
O
O
+ HS
N N
+ HS-
N N
-
O
O
pH =9.5 pH =10.1
O
O N N
S
O
N N
N N
S
O N N
O
O
O O
6.00
6.00
mAU
4.00
4.00 2.00
mAU
SPB
2.00 SDB
0.00 10.00
15.00
0.00 min 18.00 10.00
Figure 5
min
15.00
18.00
Fig.6