- Email: [email protected]
Disulfide stress in carbon monoxide poisoning
CLB-09343; No. of pages: 5; 4C: Clinical Biochemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Disulfide stress in carbon monoxide poisoning Merve Ergin a,⁎, Mustafa Caliskanturk b, Almila Senat c, Onur Akturk a, Ozcan Erel c a b c
Department of Biochemistry, 25 Aralik State Hospital, Gaziantep, Turkey Department of Emergency Medicine, 25 Aralik State Hospital, Gaziantep, Turkey Department of Biochemistry, Yildirim Beyazit University, Faculty of Medicine, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 30 March 2016 Received in revised form 25 July 2016 Accepted 28 July 2016 Available online xxxx Keywords: Carbon monoxide poisoning Disulfide Oxidative stress Thiol Thiol-disulfide homeostasis
a b s t r a c t Objectives: Carbon monoxide (CO) remains the most common cause of lethal poisoning around the world. The purpose of this study was to investigate the homeostasis between thiol-disulfide couples and to evaluate oxidative status comprehensively in acute CO poisoning, using new parameters along with other well-known oxidant-antioxidant molecules. Design and methods: This case study consisted of 43 subjects who were diagnosed with carbon monoxide poisoning and 35 healthy individuals who were used as controls. Thiol-disulfide paired tests were examined in both groups using the method developed recently. Results: Patients with CO poisoning had significantly higher levels of serum disulfide than the control patients (20.7 ± 5.03 versus 16.43 ± 3.97, p = 0.001). Native thiol and total thiol levels were lower in the CO patient group than in the control group (p b 0.001, for each variable). The disulfide/native thiol ratios and disulfide/total thiol ratios were significantly higher, while native thiol/total thiol ratios were significantly lower, in patients with acute CO poisoning than in the healthy controls (p b 0.001, for all ratios). The disulfide/ native ratios were negatively correlated with both total antioxidant response and paraoxonase and arylesterase values and were positively correlated with total oxidant status and ceruloplasmin values (p b 0.05, for all correlations). Conclusions: Excessive disulfide levels and their related ratios were found in CO poisoning patients. In particular, the disulfide/native thiol ratio was identified as an indicator for overall oxidative status. Among CO poisoning patients, the thiol-disulfide balance was found to be impaired. Therefore, the disruption of thiol-disulfide homeostasis might be involved in CO toxicity. © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
1. Introduction Carbon monoxide (CO) poisoning that results in injury or death is the cause of more than half of poisonings worldwide [1]. The recognition of CO poisoning proves to be challenging because of CO poisoning's nonspecific signs and symptoms [2]; therefore, the true morbidity and mortality rates are considered to be higher due to the misdiagnosis or non-recognition of CO poisoning [1]. CO poisoning is called the “silent killer,” owing to its odorless, colorless, and non-irritating properties [3]. CO is produced by incomplete combustion of carbon-containing compounds. Motor vehicle exhaust gases, forest fires, gas line leakage, insufficient ventilation of flame-based heaters, and industrial paints are all common sources of CO [4]. The potential mechanisms of CO toxicity depend on the hemoproteins, cytochrome oxidase, and cytochrome p450 systems [5]. Formation
⁎ Corresponding author at: Department of Biochemistry, Gaziantep 25 Aralik State Hospital, Gaziantep, Turkey. E-mail address: [email protected] (M. Ergin).
of carboxyhemoglobin (COHb) results in tissue hypoxia and ischemia. Impaired perfusion and CO-related cellular damage generate CO toxicity [6]. CO has directly toxic effects in the electron transport chain of mitochondria via binding cytochrome oxidase. This disturbance in the respiratory chain triggers oxidative stress and decreases glutathione levels [7]. Inhibition of the mitochondrial enzymes leads to lipid peroxidation on membranes [6]. Moreover, CO also binds platelets' hemoproteins, inducing nitric oxide (NO) release. Enhanced NO generates peroxynitrites and results in nitrosative stress [5]. Intravascularly, CO induces leukocyte sequestration, platelet-neutrophil aggregation, and neutrophil degranulation. Therefore, CO causes free radical production, apoptosis, and lipid peroxidation that results in endothelial dysfunction [4]. The perturbation of the cell redox balance occurs when reactive oxygen species are generated excessively, and antioxidants are not able to counteract this overproduction [8]. In the normal oxidative metabolism, reactive oxygen species are produced by the mitochondrial electron transport chain, and oxygen generation increases by the time electron transport is inhibited [9]. Thiols are known to be the most important and essential antioxidant buffers, which interact with almost all physiological oxidants [8]. Low antioxidant capacity and accompanying
http://dx.doi.org/10.1016/j.clinbiochem.2016.07.019 0009-9120/© 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: M. Ergin, et al., Disulfide stress in carbon monoxide poisoning, Clin Biochem (2016), http://dx.doi.org/10.1016/ j.clinbiochem.2016.07.019
2
M. Ergin et al. / Clinical Biochemistry xxx (2016) xxx–xxx
oxidative stress results from the decreased glutathione and oxidized glutathione ratio (GSH/GSSG) in the intracellular environment [10]. Thiols exist in proteins, including albumin and other thiolated proteins, or in low molecular-weight molecules, such as glutathione and homocysteine [10]. Groups of thiol proteins go through oxidation reactions when oxygen molecules are present, and the proteins turn into reversible forms called disulfide bridges (–S–S–). This form can be reduced to thiols again [11]. Thus, thiol-disulfide homeostasis occurs. The thiol-disulfide couple maintains a homeostatic intracellular and tissue redox status [12]. Dynamic thiol-disulfide homeostasis acts in cellular signal mechanisms, antioxidant defense, detoxification, apoptosis, inflammation, and immune response [13]. In addition, impaired thiol-disulfide status is implicated in many diseases [14,15]. Until recently, just one side of the thiol-disulfide balance was measured. However, using the latest testing methods, both sides of the balance can be detected, and the thiol-disulfide status can be completely evaluated [12]. The primary aim of this study was to examine thioldisulfide homeostasis, which performs a crucial role in CO toxicity, through thiol-disulfide paired tests. A secondary purpose of this study was evaluating the oxidative status of patients with CO poisoning through new parameters along with well-known oxidant-antioxidant parameters, including total antioxidant response (TAR), total oxidant status (TOS), paraoxonase (PON), arylesterase (ARES), and ceruloplasmin. Currently, there are no other studies related to thiol-disulfide exchanges in acute CO poisoning, and this study comprises the first report in this area.
Serum thiol-disulfide pair tests were measured through a novel spectrophotometric method [12] with an automated analyzer (Roche, cobas 501, Mannheim, Germany). In this method, disulfide bridges were reduced to free thiols via the reductant sodium borohydride. The remaining sodium borohydride was exhausted using formaldehyde. Then, the reduced thiol groups and existing native thiols were analyzed with 5, 5′-dithiobis-(2-nitrobenzoic) acid (DTNB). This resulting measurement gave the total thiol amounts. The disulfide amounts were calculated as half of the subtraction of native thiol from total thiol concentrations. Once the native thiol levels, total thiol levels, and disulfide amounts were determined, the disulfide/native thiol [(–S–S–)/(–SH)], disulfide/total thiol [(–S–S–)/(–SH + –S–S–)], and native thiol/total thiol [(–SH)/(–SH– + –S–S–)] ratios were calculated. Total antioxidant response, total oxidant status, and serum ceruloplasmin levels were determined by the automated methods described by Erel [16–18]. PON and ARES serum activity were measured by using commercially available kits (Rel Assay Diagnostics, Gaziantep, Turkey). Albumin and total protein levels were detected with commercially available assay kits (Roche, Mannheim, Germany) with an autoanalyzer (cobas 501, Roche, Mannheim, Germany). COHb level measurements were performed in a blood gas analyzer (Rapid point500, Siemens, Munich, Germany). Normobaric oxygen treatment using 100% oxygen was provided to all patients with acute CO poisoning, and none of the participants received hyperbaric oxygen therapy. 2.2. Statistical analysis
2. Materials and methods 2.1. Study protocol Forty-three patients (20 male, 23 female) with acute CO poisoning when admitted to the emergency department were enrolled in the study. Diagnosis of acute CO poisoning was based on the patients' history, clinical and laboratory findings, and patients' COHb levels [3]. Subjects were excluded from the study if they exhibited ischemic or hemorrhagic stroke, central nervous system disorders, head trauma, or other known probable unconsciousness causes, were suffering from any existing systemic, infectious, or inflammatory diseases, such as diabetes mellitus, cardiac disorders, anemia, hypertension, or showed evidence of any renal, hepatic, or gastrointestinal diseases. Additionally, all patients who were smokers, abused alcohol, or took any medications were excluded from the study. The control group comprised 35 healthy individuals (14 male, 21 female): a detailed medical history was taken and a normal physical examination performed for each subject. None of the subjects was taking vitamin supplements. Study groups were matched in terms of age and sex. The study protocol was approved by the local ethics committee, and written informed consents were obtained from all participants prior to involvement in the study. Venous blood samples were taken via venipuncture from all of the subjects and drawn into tubes. Specimens were processed by centrifugation at 1800g for 10 min. After the separation, serum samples were instantly frozen and kept at −80 °C until testing.
SPSS software version 22.0 (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analysis. Data distribution was examined by both visual (histograms, probability plots) and statistical methods (the Kolmogorov-Smirnov test and the Shapiro-Wilk test). As the variables showed normal distribution, independent sample t tests were applied to compare the parameters between groups using mean and standard deviation. Correlation analyses were performed using Pearson's correlation co-efficient. In all analyses, the outcomes were considered statistically significant if p value was b0.05. 3. Results The subjects' demographic and clinical characteristics appear in Table 1. There were no statistically significant differences between the groups in terms of age and sex. Serum albumin and total protein levels were similar between the two groups, and the differences between mean albumin and total protein levels were not statistically significant in the two groups (p N 0.05). In all patients, CO exposure occurred as a result of defective heating systems. There was no loss of consciousness when patients arrived at the hospital. In addition, no mortality occurred. Table 2 and Fig. 1 indicate the thiol-disulfide profiles of all participants. In the acute CO poisoning study group, native and total thiol levels were significantly lower than in the control group (p b 0.001, for both). When the two groups were compared, based on disulfide levels, there was a significant difference between the groups (p =
Table 1 Clinical characteristics and some laboratory findings of the study population.
Age (year) Gender (male/female) Albumin (g/dl) Total protein (g/dl) COHb (%)
CO poisoning (n = 43)
Control group (n = 35)
p Value
34.56 ± 19.05 20/23 4.41 ± 0.47 7.38 ± 0.63 19.68 ± 6.94
33.83 ± 12.53 14/21 4.53 ± 0.24 7.37 ± 0.36 0.89 ± 0.86
NS⁎ NS NS NS p b 0.001
Values are mean ± SD. p b 0.05 was accepted as statistically significant. CO, carbon monoxide; COHb, carboxyhemoglobin. ⁎ NS, non-significant.
Please cite this article as: M. Ergin, et al., Disulfide stress in carbon monoxide poisoning, Clin Biochem (2016), http://dx.doi.org/10.1016/ j.clinbiochem.2016.07.019
M. Ergin et al. / Clinical Biochemistry xxx (2016) xxx–xxx
3
Table 2 Thiol-disulfide profiles and certain oxidant-antioxidant parameters of study group. Parameters
CO poisoning n = 43
Control group n = 35
p Value⁎
Native thiol, μmol/L Total thiol, μmol/L Disulfide, μmol/L Disulfide/native thiol ratio, % Disulfide/total thiol ratio, % Native thiol/total thiol ratio, % Total antioxidant response, mmol Trolox eq/L Total oxidant status, μmol H2O2 eq/L Paraoxonase, U/L Arylesterase, KU/L Ceruloplasmin, U/L
344.29 ± 62.29 385.71 ± 66.92 20.7 ± 5.03 6.08 ± 1.20 5.35 ± 0.68 89.28 ± 2.36 0.97 ± 0.22 4.55 ± 1.33 125.3 ± 26.22 164.77 ± 35.36 94.57 ± 17.06
475 ± 49.01 507.87 ± 50.54 16.43 ± 3.97 3.51 ± 0.96 3.27 ± 0.83 93.45 ± 1.67 1.14 ± 0.26 1.97 ± 0.46 199.5 ± 40.21 186.93 ± 41.24 86.19 ± 11.8
p b 0.001 p b 0.001 p = 0.001 p b 0.001 p b 0.001 p b 0.001 p = 0.006 p b 0.001 p b 0.001 p = 0.009 p = 0.008
Results were expressed as mean ± SD. CO, carbon monoxide. ⁎ p Value b 0.05 considered significant.
0.001), and disulfide levels were higher in the CO patients than in the healthy subjects. The disulfide/native thiol ratios and disulfide/total thiol ratios were significantly higher and native/total thiol ratios were significantly lower in patients with acute CO poisoning than in the healthy controls (p b 0.001, for all ratios). TAR levels, and PON and ARES activity were significantly decreased in patients with acute CO poisoning compared with the control group (Table 2). TOS and ceruloplasmin levels of CO patients were found to be significantly higher than those of healthy individuals (Table 2). When the relationships with thiol-disulfide tests and other parameters were evaluated, as seen in Table 3, there were negative correlations between the native and total thiol levels, and TAR, PON, and ARES values, but there were positive correlations between native and total thiol amounts, and TOS and ceruloplasmin levels. Additionally, the disulfide/native thiol and disulfide/total thiol ratios significantly and negatively correlated with TAR, PON, and ARES levels, while they significantly and positively correlated with TOS and ceruloplasmin values. Significant relationships were also found between the native thiol/ total thiol ratios and other oxidant-antioxidant variables. 4. Discussion Sulfur metabolism, which includes cysteine, glutathione and homocysteine, is being researched as a potential indicator of overall health status and disease risk [19]. Modulation of the redox system controls cellular functions. The cysteine in proteins gets involved in redox reactions, such as thiol-disulfide couples, and is vulnerable to oxidoreduction reactions [10]. Exchanges in the state of redox pairs affect signaling mechanisms, and the dynamic thiol-disulfide balance has a central role within the organism [12]. Alterations in the thiol-disulfide balance contributes to the response of cells to inflammation, oxidative stress, detoxification, and apoptosis [13,20]. Therefore, impaired thiol-disulfide homeostasis has been implicated in a broad range of diseases [21,22]. The mechanism underlying the toxicity of CO is not clearly understood, and the pathology of CO poisoning is a noticeably complex mechanism [6]. CO leads to hypoxia through the formation of COHb and causes direct damage at a cellular level. Binding other hemecontaining proteins impairs mitochondrial function, increases the release of NO and produces peroxynitrite. These effects result in oxidative stress, lipid peroxidation and apoptosis [4]. To the best of our knowledge, no prior research report evaluating the role of thioldisulfide homeostasis in acute CO poisoning has been published. As can be seen in Table 2, native and total thiol levels were lower and disulfide concentrations were higher in patients with CO poisoning than in healthy controls. This status established that the thiol-disulfide balance shifted to disulfide bond formation, which indicated oxidative action. Although there were no differences in albumin and total protein levels between the two groups, native and total thiol levels were
lower in the CO patient group. These results revealed the reduction of thiols through interaction with oxidants. In addition, when CO binds cytochrome, oxidase glutathione levels decrease [3]. Therefore, diminished glutathione levels must effect thiols. In addition, enhanced disulfide/total thiol and disulfide/native thiol ratios [–S– S–/(–SH + –S–S–) and (–S–S–/–SH)], and decreased native thiol/ total thiol ratios [SH/(–SH + –S–S–)] indicated that the thioldisulfide homeostasis was impaired in acute CO poisoning. According to these results, it is very likely that there was disulfide stress in acute CO poisoning. Furthermore, the decrease in TAR, PON, and ARES levels and the increase in TOS and ceruloplasmin values demonstrated the imbalance between oxidant and antioxidant statuses. Significant associations between thiol-disulfide tests and other assessed oxidant-antioxidant molecules strengthened these results. There is growing evidence from multiple studies identifying oxidative stress as a main culprit in the tissue injury found in many human diseases [8]. Therefore, it can be suggested that oxidative stress is one of the major factors in CO toxicity [6]. Kavakli et al. found TOS levels and the oxidative stress index (OSI) higher in patients with CO poisoning than in controls [23]. After treatment, Kavakli et al. observed significantly decreased TOS and OSI levels in comparison with patients' admission levels. Those outcomes paralleled our findings of enhanced TOS levels, disulfide amounts, and increased disulfide/native thiol and disulfide/total thiol ratios. Zengin et al. compared a CO poisoning study group with healthy controls to assess their antioxidant levels [24]. Those researchers obtained significantly lower levels of –SH, PON, and ARES, and elevated levels of ceruloplasmin on admission in patients with CO poisoning.
Fig. 1. Disulfide/native thiol and disulfide/total thiol ratios between the study groups.
Please cite this article as: M. Ergin, et al., Disulfide stress in carbon monoxide poisoning, Clin Biochem (2016), http://dx.doi.org/10.1016/ j.clinbiochem.2016.07.019
4
M. Ergin et al. / Clinical Biochemistry xxx (2016) xxx–xxx
Table 3 Relationships between thiol-disulfide tests and other oxidant-antioxidant variables. n = 78
Native thiol
Total thiol
Disulfide
Disulfide/native thiol
Disulfide/total thiol
Native thiol/total thiol
Total antioxidant response
r = 0.30 p = 0.018 r = −0.48 p b 0.001 r = 0.32 p = 0.014 r = 0.29 p = 0.012 r = −0.38 p = 0.001
r = 0.28 p = 0.031 r = −0.44 p b 0.001 r = 0.28 p = 0.035 r = 0.27 p = 0.018 r = −0.36 p = 0.001
r = −0.16 p N 0.05 r = 0.30 p = 0.009 r = −0.31 p = 0.018 r = −0.13 p N 0.05 r = 0.12 p N 0.05
r = −0.27 p = 0.028 r = 0.52 p b 0.001 r = −0.36 p = 0.006 r = −0.26 p = 0.026 r = 0.30 p = 0.008
r = −0.27 p = 0.028 r = 0.52 p b 0.001 r = −0.37 p = 0.004 r = −0.26 p = 0.023 r = 0.31 p = 0.007
r = 0.27 p = 0.028 r = −0.52 p b 0.001 r = 0.37 p = 0.004 r = 0.26 p = 0.023 r = −0.31 p = 0.007
Total oxidant status Paraoxonase Arylesterase Ceruloplasmin
The r value is the Pearson correlation coefficient. The p value is significance.
That decreased antioxidant status was in harmony with our diminished TAR, PON, ARES, and thiol results, while the increase in ceruloplasmin found in Zengin et al. matched the results of our study. Impairment of mitochondrial function, oxidative stress, and lipid peroxidation comprises a vicious cycle in CO poisoning [6]. For example, Wang et al. examined the time-dependent changes in malondialdehyde (MDA), which is known as a lipid peroxidation marker, and glutathione, glutathione peroxidase, and glutathione reductase levels [25]. Increased MDA levels and decreased activity of glutathione reductase and glutathione peroxidase were found [25]. At first, investigators observed increased GSH levels, but then they found decreased levels of GSH. Similar to those findings, Yavuz et al. found increased MDA levels in the CO intoxication group and decreased MDA levels in the treatment group in an experimental model [26]. In addition, that study found decreased activity of glutathione peroxidase in the CO intoxication group and increased activities of glutathione peroxidase were found in the treatment group [26]. Moreover, Zang et al. obtained decreased ratios in reduced to oxidized glutathione (GSH/GSSG) within mitochondria after CO exposure [27]. Despite the fact that glutathione is the primarily abundant thiol component within the cellular medium, measured plasma GSH concentration constitutes a minor amount of the plasma thiol pool. Thus, the recently developed method allowed complete evaluation of the homeostatic redox environment [12]. N-acetylcysteine (NAC) is a precursor in the formation of glutathione, so it is capable of reinforcing the hepatic glutathione stores. Due to the sulfhydryl group in its structure, NAC presents antioxidant effects [28]. In a single case report, NAC and a xanthine oxidase inhibitor were applied to treat CO poisoning, and the patient achieved a full recovery [29]. Likewise, a recent experimental model has shown that NAC reduced the damage of CO exposure in brain and lung tissues [30]. Hydrogen sulfide (H2S) is an important sulfur compound that reacts with alcohols to form thiols. H2S has been regarded as a novel gaseous transmitter within the central nervous system, particularly as it presents both antioxidant and anti-apoptotic properties [31]. A recent experimental study demonstrated that H2S depressed the oxidative damage induced by CO poisoning and lessened apoptosis [32]. Additionally, Yu et al. speculated that H2S may be a novel therapy for acute CO poisoning [31]. 5. Conclusions Thiol-disulfide homeostasis is impaired in acute CO poisoning. The enhanced disulfide/native thiol ratios and disulfide/total thiol ratios found in this study reflected the extreme oxidation that occurs in CO poisoning. In this study, the relationship with the thiol-disulfide profile and other well-known oxidant-antioxidant parameters supported this fact. According to these research findings, the disulfide/native thiol ratio can be used as an indicator for oxidative stress, and disulfide stress mentioned as a factor in oxidative stress. Further extensive studies, evaluating the homeostasis between thiol-disulfide couples, may provide progressions in assessing the CO toxicity.
Transparency document The Transparency document associated with this article can be found, in online version.
References [1] J.A. Raub, M. Mathieu-Nolf, N.B. Hampson, S.R. Thom, Carbon monoxide poisoning—a public health perspective, Toxicology 145 (1) (2000) 1–14. [2] L. Wu, R. Wang, Carbon monoxide: endogenous production, physiological functions, and pharmacological applications, Pharmacol. Rev. 57 (4) (2005) 585–630, http:// dx.doi.org/10.1124/pr.57.4.3. [3] G. Lippi, G. Rastelli, T. Meschi, L. Borghi, G. Cervellin, Pathophysiology, clinics, diagnosis and treatment of heart involvement in carbon monoxide poisoning, Clin. Biochem. 45 (16–17) (2012) 1278–1285, http://dx.doi.org/10.1016/j.clinbiochem. 2012.06.004. [4] J.A. Guzman, Carbon monoxide poisoning, Crit. Care Clin. 28 (4) (2012) 537–548, http://dx.doi.org/10.1016/j.ccc.2012.07.007. [5] L.K. Weaver, Clinical practice. Carbon monoxide poisoning, N. Engl. J. Med. 360 (12) (2009) 1217–1225, http://dx.doi.org/10.1056/NEJMcp0808891. [6] S. Akyol, S. Erdogan, N. Idiz, S. Celik, M. Kaya, F. Ucar, S. Dane, O. Akyol, The role of reactive oxygen species and oxidative stress in carbon monoxide toxicity: an indepth analysis, Redox Rep. 19 (5) (2014) 180–189, http://dx.doi.org/10.1179/ 1351000214Y.0000000094. [7] D. Taskiran, T. Nesil, K. Alkan, Mitochondrial oxidative stress in female and male rat brain after ex vivo carbon monoxide treatment, Hum. Exp. Toxicol. 26 (8) (2007) 645–651, http://dx.doi.org/10.1177/0960327107076882. [8] A.M. Pisoschi, A. Pop, The role of antioxidants in the chemistry of oxidative stress: a review, Eur. J. Med. Chem. 97 (2015) 55–74, http://dx.doi.org/10.1016/j.ejmech. 2015.04.040. [9] T. Kalogeris, Y. Bao, R.J. Korthuis, Mitochondrial reactive oxygen species: a double edged sword in ischemia/reperfusion vs preconditioning, Redox Biol. 2 (2014) 702–714, http://dx.doi.org/10.1016/j.redox.2014.05.006. [10] K.Q. de Andrade, F.A. Moura, J.M. Dos Santos, O.R. de Araujo, J.C. de Farias Santos, M.O. Goulart, Oxidative stress and inflammation in hepatic diseases: therapeutic possibilities of N-acetylcysteine, Int. J. Mol. Sci. 16 (12) (2015) 30269–30308, http://dx.doi.org/10.3390/ijms161226225. [11] D.P. Jones, Y. Liang, Measuring the poise of thiol/disulfide couples in vivo, Free Radical Biol. Med. 47 (10) (2009) 1329–1338, http://dx.doi.org/10.1016/j.freeradbiomed. 2009.08.021. [12] O. Erel, S. Neselioglu, A novel and automated assay for thiol/disulphide homeostasis, Clin. Biochem. 47 (18) (2014) 326–332, http://dx.doi.org/10.1016/j.clinbiochem. 2014.09.026. [13] M.L. Circu, T.Y. Aw, Reactive oxygen species, cellular redox systems, and apoptosis, Free Radical Biol. Med. 48 (6) (2010) 749–762, http://dx.doi.org/10. 1016/j.freeradbiomed.2009.12.022. [14] H. Kundi, I. Ates, E. Kiziltunc, M. Cetin, H. Cicekcioglu, S. Neselioglu, O. Erel, E. Ornek, A novel oxidative stress marker in acute myocardial infarction; thiol/disulphide homeostasis, Am. J. Emerg. Med. 33 (11) (2015) 1567–1571, http://dx.doi.org/10. 1016/j.ajem.2015.06.016. [15] S. Ozler, O. Erel, E. Oztas, A.O. Ersoy, M. Ergin, A. Sucak, S. Neselioglu, D. Uygur, N. Danisman, Serum thiol/disulphide homeostasis in preeclampsia, Hypertens. Pregnancy 34 (4) (2015) 474–485, http://dx.doi.org/10.3109/10641955.2015.1077859. [16] O. Erel, A novel automated method to measure total antioxidant response against potent free radical reactions, Clin. Biochem. 37 (2) (2004) 112–119. [17] O. Erel, A new automated colorimetric method for mesuring total oxidant status, Clin. Biochem. 38 (12) (2005) 1103–1111. [18] O. Erel, Automated measurement of serum ferroxidase activity, Clin. Chem. 44 (11) (1998) 2313–2319. [19] W.A. Kleinman, J.P. Richie Jr., Status of glutathione and other thiols and disulfides in human plasma, Biochem. Pharmacol. 60 (1) (2000) 19–29. [20] I. Rahman, S.K. Biswas, L.A. Jimenez, M. Torres, H. Forman, Glutathione, stress responses, and redox signaling in lung inflammation, Antioxid. Redox Signaling 7 (1–2) (2005) 42–59.
Please cite this article as: M. Ergin, et al., Disulfide stress in carbon monoxide poisoning, Clin Biochem (2016), http://dx.doi.org/10.1016/ j.clinbiochem.2016.07.019
M. Ergin et al. / Clinical Biochemistry xxx (2016) xxx–xxx [21] M. Ergin, B.D. Cendek, S. Neselioglu, A.F. Avsar, O. Erel, Dynamic thiol-disulfide homeostasis in hyperemesis gravidarum, J. Perinatol. 35 (10) (2015) 788–792, http://dx.doi.org/10.1038/jp.2015.81. [22] I. Ates, M. Kaplan, B. Inan, M. Alısık, O. Erel, N. Yilmaz, S. Guler, How does thiol/disulfide homeostasis change in prediabetic patients? Diabetes Res. Clin. Pract. 110 (2) (2015) 166–171. [23] H.S. Kavakli, O. Erel, O. Delice, G. Gormez, S. Isikoglu, F. Tanriverdi, Oxidative stress increases in carbon monoxide poisoning patients, Hum. Exp. Toxicol. 30 (2) (2011) 160–164, http://dx.doi.org/10.1177/0960327110388539. [24] S. Zengin, A B, S. Karta, B. Can, M. Orkmez, A. Taskin, U. Lok, B. Gulen, C. Yildirim, S. Taysi, An assessment of antioxidant status in patients with carbon monoxide poisoning, World J. Emerg. Med. 5 (2) (2014) 91–95, http://dx.doi.org/10.5847/wjem. j.1920-8642.2014.02.002. [25] P. Wang, T. Zeng, C.-L. Zhang, X.-C. Gao, Z. Liu, K.-Q. Xie, Z.-F. Chi, Lipid peroxidation was involved in the memory impairment of carbon monoxide-induced delayed neuron damage, Neurochem. Res. 34 (7) (2009) 1293–1298. [26] Y. Yavuz, H. Mollaoglu, Y. Yurumez, K. Ucok, L. Duran, K. Tunay, L. Akgun, Therapeutic effect of magnesium sulphate on carbon monoxide toxicity-mediated brain lipid peroxidation, Eur. Rev. Med. Pharmacol. Sci. 17 (Suppl. 1) (2013) 28–33.
5
[27] J. Zhang, C.A. Piantadosi, Mitochondrial oxidative stress after carbon monoxide hypoxia in the rat brain, J. Clin. Invest. 90 (4) (1992) 1193–1199, http://dx.doi.org/10. 1172/JCI115980. [28] G.F. Rushworth, I.L. Megson, Existing and potential therapeutic uses for Nacetylcysteine: the need for conversion to intracellular glutathione for antioxidant benefits, Pharmacol. Ther. 141 (2) (2014) 150–159, http://dx.doi.org/10.1016/j. pharmthera.2013.09.006. [29] R.J. Howard, D.R. Blake, H. Pall, A. Williams, I.D. Green, Allopurinol/N-acetylcysteine for carbon monoxide poisoning, Lancet 2 (8559) (1987) 628–629. [30] Z. Kekec, G. Seydaoglul, H. Sever, F. Ozturk, The effect of antioxidants (Nacetylcysteine and melatonin) on hypoxia due to carbonmonoxide poisoning, Bratisl. Lek. Listy 111 (4) (2009) 189–193. [31] Y.-P. Yu, Z.-G. Li, D.-Z. Wang, X. Zhan, J.-H. Shao, Hydrogen sulfide as an effective and specific novel therapy for acute carbon monoxide poisoning, Biochem. Biophys. Res. Commun. 404 (1) (2011) 6–9. [32] J. Zhang, H. Wu, Y. Zhao, H. Zu, Therapeutic effects of hydrogen sulfide in treating delayed encephalopathy after acute carbon monoxide poisoning, Am. J. Ther. (2015) http://dx.doi.org/10.1097/MJT.0000000000000290.
Please cite this article as: M. Ergin, et al., Disulfide stress in carbon monoxide poisoning, Clin Biochem (2016), http://dx.doi.org/10.1016/ j.clinbiochem.2016.07.019
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Disulfide stress in carbon monoxide poisoning Merve Ergin a,⁎, Mustafa Caliskanturk b, Almila Senat c, Onur Akturk a, Ozcan Erel c a b c
Department of Biochemistry, 25 Aralik State Hospital, Gaziantep, Turkey Department of Emergency Medicine, 25 Aralik State Hospital, Gaziantep, Turkey Department of Biochemistry, Yildirim Beyazit University, Faculty of Medicine, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 30 March 2016 Received in revised form 25 July 2016 Accepted 28 July 2016 Available online xxxx Keywords: Carbon monoxide poisoning Disulfide Oxidative stress Thiol Thiol-disulfide homeostasis
a b s t r a c t Objectives: Carbon monoxide (CO) remains the most common cause of lethal poisoning around the world. The purpose of this study was to investigate the homeostasis between thiol-disulfide couples and to evaluate oxidative status comprehensively in acute CO poisoning, using new parameters along with other well-known oxidant-antioxidant molecules. Design and methods: This case study consisted of 43 subjects who were diagnosed with carbon monoxide poisoning and 35 healthy individuals who were used as controls. Thiol-disulfide paired tests were examined in both groups using the method developed recently. Results: Patients with CO poisoning had significantly higher levels of serum disulfide than the control patients (20.7 ± 5.03 versus 16.43 ± 3.97, p = 0.001). Native thiol and total thiol levels were lower in the CO patient group than in the control group (p b 0.001, for each variable). The disulfide/native thiol ratios and disulfide/total thiol ratios were significantly higher, while native thiol/total thiol ratios were significantly lower, in patients with acute CO poisoning than in the healthy controls (p b 0.001, for all ratios). The disulfide/ native ratios were negatively correlated with both total antioxidant response and paraoxonase and arylesterase values and were positively correlated with total oxidant status and ceruloplasmin values (p b 0.05, for all correlations). Conclusions: Excessive disulfide levels and their related ratios were found in CO poisoning patients. In particular, the disulfide/native thiol ratio was identified as an indicator for overall oxidative status. Among CO poisoning patients, the thiol-disulfide balance was found to be impaired. Therefore, the disruption of thiol-disulfide homeostasis might be involved in CO toxicity. © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
1. Introduction Carbon monoxide (CO) poisoning that results in injury or death is the cause of more than half of poisonings worldwide [1]. The recognition of CO poisoning proves to be challenging because of CO poisoning's nonspecific signs and symptoms [2]; therefore, the true morbidity and mortality rates are considered to be higher due to the misdiagnosis or non-recognition of CO poisoning [1]. CO poisoning is called the “silent killer,” owing to its odorless, colorless, and non-irritating properties [3]. CO is produced by incomplete combustion of carbon-containing compounds. Motor vehicle exhaust gases, forest fires, gas line leakage, insufficient ventilation of flame-based heaters, and industrial paints are all common sources of CO [4]. The potential mechanisms of CO toxicity depend on the hemoproteins, cytochrome oxidase, and cytochrome p450 systems [5]. Formation
⁎ Corresponding author at: Department of Biochemistry, Gaziantep 25 Aralik State Hospital, Gaziantep, Turkey. E-mail address: [email protected] (M. Ergin).
of carboxyhemoglobin (COHb) results in tissue hypoxia and ischemia. Impaired perfusion and CO-related cellular damage generate CO toxicity [6]. CO has directly toxic effects in the electron transport chain of mitochondria via binding cytochrome oxidase. This disturbance in the respiratory chain triggers oxidative stress and decreases glutathione levels [7]. Inhibition of the mitochondrial enzymes leads to lipid peroxidation on membranes [6]. Moreover, CO also binds platelets' hemoproteins, inducing nitric oxide (NO) release. Enhanced NO generates peroxynitrites and results in nitrosative stress [5]. Intravascularly, CO induces leukocyte sequestration, platelet-neutrophil aggregation, and neutrophil degranulation. Therefore, CO causes free radical production, apoptosis, and lipid peroxidation that results in endothelial dysfunction [4]. The perturbation of the cell redox balance occurs when reactive oxygen species are generated excessively, and antioxidants are not able to counteract this overproduction [8]. In the normal oxidative metabolism, reactive oxygen species are produced by the mitochondrial electron transport chain, and oxygen generation increases by the time electron transport is inhibited [9]. Thiols are known to be the most important and essential antioxidant buffers, which interact with almost all physiological oxidants [8]. Low antioxidant capacity and accompanying
http://dx.doi.org/10.1016/j.clinbiochem.2016.07.019 0009-9120/© 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: M. Ergin, et al., Disulfide stress in carbon monoxide poisoning, Clin Biochem (2016), http://dx.doi.org/10.1016/ j.clinbiochem.2016.07.019
2
M. Ergin et al. / Clinical Biochemistry xxx (2016) xxx–xxx
oxidative stress results from the decreased glutathione and oxidized glutathione ratio (GSH/GSSG) in the intracellular environment [10]. Thiols exist in proteins, including albumin and other thiolated proteins, or in low molecular-weight molecules, such as glutathione and homocysteine [10]. Groups of thiol proteins go through oxidation reactions when oxygen molecules are present, and the proteins turn into reversible forms called disulfide bridges (–S–S–). This form can be reduced to thiols again [11]. Thus, thiol-disulfide homeostasis occurs. The thiol-disulfide couple maintains a homeostatic intracellular and tissue redox status [12]. Dynamic thiol-disulfide homeostasis acts in cellular signal mechanisms, antioxidant defense, detoxification, apoptosis, inflammation, and immune response [13]. In addition, impaired thiol-disulfide status is implicated in many diseases [14,15]. Until recently, just one side of the thiol-disulfide balance was measured. However, using the latest testing methods, both sides of the balance can be detected, and the thiol-disulfide status can be completely evaluated [12]. The primary aim of this study was to examine thioldisulfide homeostasis, which performs a crucial role in CO toxicity, through thiol-disulfide paired tests. A secondary purpose of this study was evaluating the oxidative status of patients with CO poisoning through new parameters along with well-known oxidant-antioxidant parameters, including total antioxidant response (TAR), total oxidant status (TOS), paraoxonase (PON), arylesterase (ARES), and ceruloplasmin. Currently, there are no other studies related to thiol-disulfide exchanges in acute CO poisoning, and this study comprises the first report in this area.
Serum thiol-disulfide pair tests were measured through a novel spectrophotometric method [12] with an automated analyzer (Roche, cobas 501, Mannheim, Germany). In this method, disulfide bridges were reduced to free thiols via the reductant sodium borohydride. The remaining sodium borohydride was exhausted using formaldehyde. Then, the reduced thiol groups and existing native thiols were analyzed with 5, 5′-dithiobis-(2-nitrobenzoic) acid (DTNB). This resulting measurement gave the total thiol amounts. The disulfide amounts were calculated as half of the subtraction of native thiol from total thiol concentrations. Once the native thiol levels, total thiol levels, and disulfide amounts were determined, the disulfide/native thiol [(–S–S–)/(–SH)], disulfide/total thiol [(–S–S–)/(–SH + –S–S–)], and native thiol/total thiol [(–SH)/(–SH– + –S–S–)] ratios were calculated. Total antioxidant response, total oxidant status, and serum ceruloplasmin levels were determined by the automated methods described by Erel [16–18]. PON and ARES serum activity were measured by using commercially available kits (Rel Assay Diagnostics, Gaziantep, Turkey). Albumin and total protein levels were detected with commercially available assay kits (Roche, Mannheim, Germany) with an autoanalyzer (cobas 501, Roche, Mannheim, Germany). COHb level measurements were performed in a blood gas analyzer (Rapid point500, Siemens, Munich, Germany). Normobaric oxygen treatment using 100% oxygen was provided to all patients with acute CO poisoning, and none of the participants received hyperbaric oxygen therapy. 2.2. Statistical analysis
2. Materials and methods 2.1. Study protocol Forty-three patients (20 male, 23 female) with acute CO poisoning when admitted to the emergency department were enrolled in the study. Diagnosis of acute CO poisoning was based on the patients' history, clinical and laboratory findings, and patients' COHb levels [3]. Subjects were excluded from the study if they exhibited ischemic or hemorrhagic stroke, central nervous system disorders, head trauma, or other known probable unconsciousness causes, were suffering from any existing systemic, infectious, or inflammatory diseases, such as diabetes mellitus, cardiac disorders, anemia, hypertension, or showed evidence of any renal, hepatic, or gastrointestinal diseases. Additionally, all patients who were smokers, abused alcohol, or took any medications were excluded from the study. The control group comprised 35 healthy individuals (14 male, 21 female): a detailed medical history was taken and a normal physical examination performed for each subject. None of the subjects was taking vitamin supplements. Study groups were matched in terms of age and sex. The study protocol was approved by the local ethics committee, and written informed consents were obtained from all participants prior to involvement in the study. Venous blood samples were taken via venipuncture from all of the subjects and drawn into tubes. Specimens were processed by centrifugation at 1800g for 10 min. After the separation, serum samples were instantly frozen and kept at −80 °C until testing.
SPSS software version 22.0 (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analysis. Data distribution was examined by both visual (histograms, probability plots) and statistical methods (the Kolmogorov-Smirnov test and the Shapiro-Wilk test). As the variables showed normal distribution, independent sample t tests were applied to compare the parameters between groups using mean and standard deviation. Correlation analyses were performed using Pearson's correlation co-efficient. In all analyses, the outcomes were considered statistically significant if p value was b0.05. 3. Results The subjects' demographic and clinical characteristics appear in Table 1. There were no statistically significant differences between the groups in terms of age and sex. Serum albumin and total protein levels were similar between the two groups, and the differences between mean albumin and total protein levels were not statistically significant in the two groups (p N 0.05). In all patients, CO exposure occurred as a result of defective heating systems. There was no loss of consciousness when patients arrived at the hospital. In addition, no mortality occurred. Table 2 and Fig. 1 indicate the thiol-disulfide profiles of all participants. In the acute CO poisoning study group, native and total thiol levels were significantly lower than in the control group (p b 0.001, for both). When the two groups were compared, based on disulfide levels, there was a significant difference between the groups (p =
Table 1 Clinical characteristics and some laboratory findings of the study population.
Age (year) Gender (male/female) Albumin (g/dl) Total protein (g/dl) COHb (%)
CO poisoning (n = 43)
Control group (n = 35)
p Value
34.56 ± 19.05 20/23 4.41 ± 0.47 7.38 ± 0.63 19.68 ± 6.94
33.83 ± 12.53 14/21 4.53 ± 0.24 7.37 ± 0.36 0.89 ± 0.86
NS⁎ NS NS NS p b 0.001
Values are mean ± SD. p b 0.05 was accepted as statistically significant. CO, carbon monoxide; COHb, carboxyhemoglobin. ⁎ NS, non-significant.
Please cite this article as: M. Ergin, et al., Disulfide stress in carbon monoxide poisoning, Clin Biochem (2016), http://dx.doi.org/10.1016/ j.clinbiochem.2016.07.019
M. Ergin et al. / Clinical Biochemistry xxx (2016) xxx–xxx
3
Table 2 Thiol-disulfide profiles and certain oxidant-antioxidant parameters of study group. Parameters
CO poisoning n = 43
Control group n = 35
p Value⁎
Native thiol, μmol/L Total thiol, μmol/L Disulfide, μmol/L Disulfide/native thiol ratio, % Disulfide/total thiol ratio, % Native thiol/total thiol ratio, % Total antioxidant response, mmol Trolox eq/L Total oxidant status, μmol H2O2 eq/L Paraoxonase, U/L Arylesterase, KU/L Ceruloplasmin, U/L
344.29 ± 62.29 385.71 ± 66.92 20.7 ± 5.03 6.08 ± 1.20 5.35 ± 0.68 89.28 ± 2.36 0.97 ± 0.22 4.55 ± 1.33 125.3 ± 26.22 164.77 ± 35.36 94.57 ± 17.06
475 ± 49.01 507.87 ± 50.54 16.43 ± 3.97 3.51 ± 0.96 3.27 ± 0.83 93.45 ± 1.67 1.14 ± 0.26 1.97 ± 0.46 199.5 ± 40.21 186.93 ± 41.24 86.19 ± 11.8
p b 0.001 p b 0.001 p = 0.001 p b 0.001 p b 0.001 p b 0.001 p = 0.006 p b 0.001 p b 0.001 p = 0.009 p = 0.008
Results were expressed as mean ± SD. CO, carbon monoxide. ⁎ p Value b 0.05 considered significant.
0.001), and disulfide levels were higher in the CO patients than in the healthy subjects. The disulfide/native thiol ratios and disulfide/total thiol ratios were significantly higher and native/total thiol ratios were significantly lower in patients with acute CO poisoning than in the healthy controls (p b 0.001, for all ratios). TAR levels, and PON and ARES activity were significantly decreased in patients with acute CO poisoning compared with the control group (Table 2). TOS and ceruloplasmin levels of CO patients were found to be significantly higher than those of healthy individuals (Table 2). When the relationships with thiol-disulfide tests and other parameters were evaluated, as seen in Table 3, there were negative correlations between the native and total thiol levels, and TAR, PON, and ARES values, but there were positive correlations between native and total thiol amounts, and TOS and ceruloplasmin levels. Additionally, the disulfide/native thiol and disulfide/total thiol ratios significantly and negatively correlated with TAR, PON, and ARES levels, while they significantly and positively correlated with TOS and ceruloplasmin values. Significant relationships were also found between the native thiol/ total thiol ratios and other oxidant-antioxidant variables. 4. Discussion Sulfur metabolism, which includes cysteine, glutathione and homocysteine, is being researched as a potential indicator of overall health status and disease risk [19]. Modulation of the redox system controls cellular functions. The cysteine in proteins gets involved in redox reactions, such as thiol-disulfide couples, and is vulnerable to oxidoreduction reactions [10]. Exchanges in the state of redox pairs affect signaling mechanisms, and the dynamic thiol-disulfide balance has a central role within the organism [12]. Alterations in the thiol-disulfide balance contributes to the response of cells to inflammation, oxidative stress, detoxification, and apoptosis [13,20]. Therefore, impaired thiol-disulfide homeostasis has been implicated in a broad range of diseases [21,22]. The mechanism underlying the toxicity of CO is not clearly understood, and the pathology of CO poisoning is a noticeably complex mechanism [6]. CO leads to hypoxia through the formation of COHb and causes direct damage at a cellular level. Binding other hemecontaining proteins impairs mitochondrial function, increases the release of NO and produces peroxynitrite. These effects result in oxidative stress, lipid peroxidation and apoptosis [4]. To the best of our knowledge, no prior research report evaluating the role of thioldisulfide homeostasis in acute CO poisoning has been published. As can be seen in Table 2, native and total thiol levels were lower and disulfide concentrations were higher in patients with CO poisoning than in healthy controls. This status established that the thiol-disulfide balance shifted to disulfide bond formation, which indicated oxidative action. Although there were no differences in albumin and total protein levels between the two groups, native and total thiol levels were
lower in the CO patient group. These results revealed the reduction of thiols through interaction with oxidants. In addition, when CO binds cytochrome, oxidase glutathione levels decrease [3]. Therefore, diminished glutathione levels must effect thiols. In addition, enhanced disulfide/total thiol and disulfide/native thiol ratios [–S– S–/(–SH + –S–S–) and (–S–S–/–SH)], and decreased native thiol/ total thiol ratios [SH/(–SH + –S–S–)] indicated that the thioldisulfide homeostasis was impaired in acute CO poisoning. According to these results, it is very likely that there was disulfide stress in acute CO poisoning. Furthermore, the decrease in TAR, PON, and ARES levels and the increase in TOS and ceruloplasmin values demonstrated the imbalance between oxidant and antioxidant statuses. Significant associations between thiol-disulfide tests and other assessed oxidant-antioxidant molecules strengthened these results. There is growing evidence from multiple studies identifying oxidative stress as a main culprit in the tissue injury found in many human diseases [8]. Therefore, it can be suggested that oxidative stress is one of the major factors in CO toxicity [6]. Kavakli et al. found TOS levels and the oxidative stress index (OSI) higher in patients with CO poisoning than in controls [23]. After treatment, Kavakli et al. observed significantly decreased TOS and OSI levels in comparison with patients' admission levels. Those outcomes paralleled our findings of enhanced TOS levels, disulfide amounts, and increased disulfide/native thiol and disulfide/total thiol ratios. Zengin et al. compared a CO poisoning study group with healthy controls to assess their antioxidant levels [24]. Those researchers obtained significantly lower levels of –SH, PON, and ARES, and elevated levels of ceruloplasmin on admission in patients with CO poisoning.
Fig. 1. Disulfide/native thiol and disulfide/total thiol ratios between the study groups.
Please cite this article as: M. Ergin, et al., Disulfide stress in carbon monoxide poisoning, Clin Biochem (2016), http://dx.doi.org/10.1016/ j.clinbiochem.2016.07.019
4
M. Ergin et al. / Clinical Biochemistry xxx (2016) xxx–xxx
Table 3 Relationships between thiol-disulfide tests and other oxidant-antioxidant variables. n = 78
Native thiol
Total thiol
Disulfide
Disulfide/native thiol
Disulfide/total thiol
Native thiol/total thiol
Total antioxidant response
r = 0.30 p = 0.018 r = −0.48 p b 0.001 r = 0.32 p = 0.014 r = 0.29 p = 0.012 r = −0.38 p = 0.001
r = 0.28 p = 0.031 r = −0.44 p b 0.001 r = 0.28 p = 0.035 r = 0.27 p = 0.018 r = −0.36 p = 0.001
r = −0.16 p N 0.05 r = 0.30 p = 0.009 r = −0.31 p = 0.018 r = −0.13 p N 0.05 r = 0.12 p N 0.05
r = −0.27 p = 0.028 r = 0.52 p b 0.001 r = −0.36 p = 0.006 r = −0.26 p = 0.026 r = 0.30 p = 0.008
r = −0.27 p = 0.028 r = 0.52 p b 0.001 r = −0.37 p = 0.004 r = −0.26 p = 0.023 r = 0.31 p = 0.007
r = 0.27 p = 0.028 r = −0.52 p b 0.001 r = 0.37 p = 0.004 r = 0.26 p = 0.023 r = −0.31 p = 0.007
Total oxidant status Paraoxonase Arylesterase Ceruloplasmin
The r value is the Pearson correlation coefficient. The p value is significance.
That decreased antioxidant status was in harmony with our diminished TAR, PON, ARES, and thiol results, while the increase in ceruloplasmin found in Zengin et al. matched the results of our study. Impairment of mitochondrial function, oxidative stress, and lipid peroxidation comprises a vicious cycle in CO poisoning [6]. For example, Wang et al. examined the time-dependent changes in malondialdehyde (MDA), which is known as a lipid peroxidation marker, and glutathione, glutathione peroxidase, and glutathione reductase levels [25]. Increased MDA levels and decreased activity of glutathione reductase and glutathione peroxidase were found [25]. At first, investigators observed increased GSH levels, but then they found decreased levels of GSH. Similar to those findings, Yavuz et al. found increased MDA levels in the CO intoxication group and decreased MDA levels in the treatment group in an experimental model [26]. In addition, that study found decreased activity of glutathione peroxidase in the CO intoxication group and increased activities of glutathione peroxidase were found in the treatment group [26]. Moreover, Zang et al. obtained decreased ratios in reduced to oxidized glutathione (GSH/GSSG) within mitochondria after CO exposure [27]. Despite the fact that glutathione is the primarily abundant thiol component within the cellular medium, measured plasma GSH concentration constitutes a minor amount of the plasma thiol pool. Thus, the recently developed method allowed complete evaluation of the homeostatic redox environment [12]. N-acetylcysteine (NAC) is a precursor in the formation of glutathione, so it is capable of reinforcing the hepatic glutathione stores. Due to the sulfhydryl group in its structure, NAC presents antioxidant effects [28]. In a single case report, NAC and a xanthine oxidase inhibitor were applied to treat CO poisoning, and the patient achieved a full recovery [29]. Likewise, a recent experimental model has shown that NAC reduced the damage of CO exposure in brain and lung tissues [30]. Hydrogen sulfide (H2S) is an important sulfur compound that reacts with alcohols to form thiols. H2S has been regarded as a novel gaseous transmitter within the central nervous system, particularly as it presents both antioxidant and anti-apoptotic properties [31]. A recent experimental study demonstrated that H2S depressed the oxidative damage induced by CO poisoning and lessened apoptosis [32]. Additionally, Yu et al. speculated that H2S may be a novel therapy for acute CO poisoning [31]. 5. Conclusions Thiol-disulfide homeostasis is impaired in acute CO poisoning. The enhanced disulfide/native thiol ratios and disulfide/total thiol ratios found in this study reflected the extreme oxidation that occurs in CO poisoning. In this study, the relationship with the thiol-disulfide profile and other well-known oxidant-antioxidant parameters supported this fact. According to these research findings, the disulfide/native thiol ratio can be used as an indicator for oxidative stress, and disulfide stress mentioned as a factor in oxidative stress. Further extensive studies, evaluating the homeostasis between thiol-disulfide couples, may provide progressions in assessing the CO toxicity.
Transparency document The Transparency document associated with this article can be found, in online version.
References [1] J.A. Raub, M. Mathieu-Nolf, N.B. Hampson, S.R. Thom, Carbon monoxide poisoning—a public health perspective, Toxicology 145 (1) (2000) 1–14. [2] L. Wu, R. Wang, Carbon monoxide: endogenous production, physiological functions, and pharmacological applications, Pharmacol. Rev. 57 (4) (2005) 585–630, http:// dx.doi.org/10.1124/pr.57.4.3. [3] G. Lippi, G. Rastelli, T. Meschi, L. Borghi, G. Cervellin, Pathophysiology, clinics, diagnosis and treatment of heart involvement in carbon monoxide poisoning, Clin. Biochem. 45 (16–17) (2012) 1278–1285, http://dx.doi.org/10.1016/j.clinbiochem. 2012.06.004. [4] J.A. Guzman, Carbon monoxide poisoning, Crit. Care Clin. 28 (4) (2012) 537–548, http://dx.doi.org/10.1016/j.ccc.2012.07.007. [5] L.K. Weaver, Clinical practice. Carbon monoxide poisoning, N. Engl. J. Med. 360 (12) (2009) 1217–1225, http://dx.doi.org/10.1056/NEJMcp0808891. [6] S. Akyol, S. Erdogan, N. Idiz, S. Celik, M. Kaya, F. Ucar, S. Dane, O. Akyol, The role of reactive oxygen species and oxidative stress in carbon monoxide toxicity: an indepth analysis, Redox Rep. 19 (5) (2014) 180–189, http://dx.doi.org/10.1179/ 1351000214Y.0000000094. [7] D. Taskiran, T. Nesil, K. Alkan, Mitochondrial oxidative stress in female and male rat brain after ex vivo carbon monoxide treatment, Hum. Exp. Toxicol. 26 (8) (2007) 645–651, http://dx.doi.org/10.1177/0960327107076882. [8] A.M. Pisoschi, A. Pop, The role of antioxidants in the chemistry of oxidative stress: a review, Eur. J. Med. Chem. 97 (2015) 55–74, http://dx.doi.org/10.1016/j.ejmech. 2015.04.040. [9] T. Kalogeris, Y. Bao, R.J. Korthuis, Mitochondrial reactive oxygen species: a double edged sword in ischemia/reperfusion vs preconditioning, Redox Biol. 2 (2014) 702–714, http://dx.doi.org/10.1016/j.redox.2014.05.006. [10] K.Q. de Andrade, F.A. Moura, J.M. Dos Santos, O.R. de Araujo, J.C. de Farias Santos, M.O. Goulart, Oxidative stress and inflammation in hepatic diseases: therapeutic possibilities of N-acetylcysteine, Int. J. Mol. Sci. 16 (12) (2015) 30269–30308, http://dx.doi.org/10.3390/ijms161226225. [11] D.P. Jones, Y. Liang, Measuring the poise of thiol/disulfide couples in vivo, Free Radical Biol. Med. 47 (10) (2009) 1329–1338, http://dx.doi.org/10.1016/j.freeradbiomed. 2009.08.021. [12] O. Erel, S. Neselioglu, A novel and automated assay for thiol/disulphide homeostasis, Clin. Biochem. 47 (18) (2014) 326–332, http://dx.doi.org/10.1016/j.clinbiochem. 2014.09.026. [13] M.L. Circu, T.Y. Aw, Reactive oxygen species, cellular redox systems, and apoptosis, Free Radical Biol. Med. 48 (6) (2010) 749–762, http://dx.doi.org/10. 1016/j.freeradbiomed.2009.12.022. [14] H. Kundi, I. Ates, E. Kiziltunc, M. Cetin, H. Cicekcioglu, S. Neselioglu, O. Erel, E. Ornek, A novel oxidative stress marker in acute myocardial infarction; thiol/disulphide homeostasis, Am. J. Emerg. Med. 33 (11) (2015) 1567–1571, http://dx.doi.org/10. 1016/j.ajem.2015.06.016. [15] S. Ozler, O. Erel, E. Oztas, A.O. Ersoy, M. Ergin, A. Sucak, S. Neselioglu, D. Uygur, N. Danisman, Serum thiol/disulphide homeostasis in preeclampsia, Hypertens. Pregnancy 34 (4) (2015) 474–485, http://dx.doi.org/10.3109/10641955.2015.1077859. [16] O. Erel, A novel automated method to measure total antioxidant response against potent free radical reactions, Clin. Biochem. 37 (2) (2004) 112–119. [17] O. Erel, A new automated colorimetric method for mesuring total oxidant status, Clin. Biochem. 38 (12) (2005) 1103–1111. [18] O. Erel, Automated measurement of serum ferroxidase activity, Clin. Chem. 44 (11) (1998) 2313–2319. [19] W.A. Kleinman, J.P. Richie Jr., Status of glutathione and other thiols and disulfides in human plasma, Biochem. Pharmacol. 60 (1) (2000) 19–29. [20] I. Rahman, S.K. Biswas, L.A. Jimenez, M. Torres, H. Forman, Glutathione, stress responses, and redox signaling in lung inflammation, Antioxid. Redox Signaling 7 (1–2) (2005) 42–59.
Please cite this article as: M. Ergin, et al., Disulfide stress in carbon monoxide poisoning, Clin Biochem (2016), http://dx.doi.org/10.1016/ j.clinbiochem.2016.07.019
M. Ergin et al. / Clinical Biochemistry xxx (2016) xxx–xxx [21] M. Ergin, B.D. Cendek, S. Neselioglu, A.F. Avsar, O. Erel, Dynamic thiol-disulfide homeostasis in hyperemesis gravidarum, J. Perinatol. 35 (10) (2015) 788–792, http://dx.doi.org/10.1038/jp.2015.81. [22] I. Ates, M. Kaplan, B. Inan, M. Alısık, O. Erel, N. Yilmaz, S. Guler, How does thiol/disulfide homeostasis change in prediabetic patients? Diabetes Res. Clin. Pract. 110 (2) (2015) 166–171. [23] H.S. Kavakli, O. Erel, O. Delice, G. Gormez, S. Isikoglu, F. Tanriverdi, Oxidative stress increases in carbon monoxide poisoning patients, Hum. Exp. Toxicol. 30 (2) (2011) 160–164, http://dx.doi.org/10.1177/0960327110388539. [24] S. Zengin, A B, S. Karta, B. Can, M. Orkmez, A. Taskin, U. Lok, B. Gulen, C. Yildirim, S. Taysi, An assessment of antioxidant status in patients with carbon monoxide poisoning, World J. Emerg. Med. 5 (2) (2014) 91–95, http://dx.doi.org/10.5847/wjem. j.1920-8642.2014.02.002. [25] P. Wang, T. Zeng, C.-L. Zhang, X.-C. Gao, Z. Liu, K.-Q. Xie, Z.-F. Chi, Lipid peroxidation was involved in the memory impairment of carbon monoxide-induced delayed neuron damage, Neurochem. Res. 34 (7) (2009) 1293–1298. [26] Y. Yavuz, H. Mollaoglu, Y. Yurumez, K. Ucok, L. Duran, K. Tunay, L. Akgun, Therapeutic effect of magnesium sulphate on carbon monoxide toxicity-mediated brain lipid peroxidation, Eur. Rev. Med. Pharmacol. Sci. 17 (Suppl. 1) (2013) 28–33.
5
[27] J. Zhang, C.A. Piantadosi, Mitochondrial oxidative stress after carbon monoxide hypoxia in the rat brain, J. Clin. Invest. 90 (4) (1992) 1193–1199, http://dx.doi.org/10. 1172/JCI115980. [28] G.F. Rushworth, I.L. Megson, Existing and potential therapeutic uses for Nacetylcysteine: the need for conversion to intracellular glutathione for antioxidant benefits, Pharmacol. Ther. 141 (2) (2014) 150–159, http://dx.doi.org/10.1016/j. pharmthera.2013.09.006. [29] R.J. Howard, D.R. Blake, H. Pall, A. Williams, I.D. Green, Allopurinol/N-acetylcysteine for carbon monoxide poisoning, Lancet 2 (8559) (1987) 628–629. [30] Z. Kekec, G. Seydaoglul, H. Sever, F. Ozturk, The effect of antioxidants (Nacetylcysteine and melatonin) on hypoxia due to carbonmonoxide poisoning, Bratisl. Lek. Listy 111 (4) (2009) 189–193. [31] Y.-P. Yu, Z.-G. Li, D.-Z. Wang, X. Zhan, J.-H. Shao, Hydrogen sulfide as an effective and specific novel therapy for acute carbon monoxide poisoning, Biochem. Biophys. Res. Commun. 404 (1) (2011) 6–9. [32] J. Zhang, H. Wu, Y. Zhao, H. Zu, Therapeutic effects of hydrogen sulfide in treating delayed encephalopathy after acute carbon monoxide poisoning, Am. J. Ther. (2015) http://dx.doi.org/10.1097/MJT.0000000000000290.
Please cite this article as: M. Ergin, et al., Disulfide stress in carbon monoxide poisoning, Clin Biochem (2016), http://dx.doi.org/10.1016/ j.clinbiochem.2016.07.019