An assessment of oxidant/antioxidant status in patients with snake envenomation
- Suat Zengin1,
- Behcet Al1,
- Pinar Yarbil2,
- Remzi Guzel1,
- Mustafa Orkmez3,
- Cuma Yildirim1,
- Seyithan Taysi3
- 1Department of Emergency Medicine, Gaziantep University School of Medicine, Gaziantep, Turkey
- 2Department of Emergency Medicine, Şehitkamil State Hospital, Gaziantep, Turkey
- 3Department of Biochemistry, Gaziantep University School of Medicine, Gaziantep, Turkey
- Correspondence to Dr Suat Zengin, Emergency Department of Medicine Faculty, Gaziantep University School of Medicine, Gaziantep 27100, Turkey;
- Received 27 September 2012
- Revised 15 November 2012
- Accepted 23 November 2012
- Published Online First 22 December 2012
Objective The aim of this study is to investigate the antioxidant status (TAS), oxidant status (TOS) and oxidative stress index (OSI) in patients with snake envenomation and to learn more about the pathophysiology of snake envenomation.
Method Between May 2009 and October 2010, 47 patients were admitted to our emergency department with snake bites, and as a control group 20 healthy volunteers were enrolled in this study. Serum, plasma, and erythrocyte components were prepared for all patients on admission and at the control after 1 month. Serum TOS/TAS levels were measured.
Results No correlation was observed among age, gender and the levels of TAS, TOS and OSI. TAS, TOS and OSI levels were higher in snake envenomation patients upon arrival at the emergency department than in the healthy control group. Upon admission, all levels of patients with snake envenomation were higher than the control levels taken after 1 month.
Conclusions Serum TAS, TOS and OSI levels increase in snake envenomation patients. The results obtained in this study indicate that the snake bite was associated with a shift to an oxidative state, and oxidative stress plays an important role in the pathophysiology of snake envenomation.
Snake bites are common in many regions of the world, particularly in rural areas, and injuries occur most often during the summer months. More than 2.5 million people are bitten by snakes each year.1 The venom of many snake species consists of carbohydrates, lipids, amines, enzymes, and non-toxic and toxic proteins.2 These substances decrease the coagulability of blood, induce bleeding and secondary effects thereof, such as hypovolaemic shock and organ damage, or induce thrombosis.1–3
Free radicals (oxidants) are defined as molecules or molecular fragments containing one or more unpaired electrons in atomic or molecular orbitals.4 Sources that form free radicals are classified into two groups: endogens (mostly mitochondrial electron transport chains) and exogens (such as cigarettes, pesticides, solvents, petrochemical products, drugs, alcohol, solar rays, stress, x-rays and heavy physical activity).4 ,5 Although they have a very short life cycle, free radicals may be harmful to organisms due to their activity levels.6
The human body has antioxidative mechanisms to overwhelm oxidants. Antioxidants are classified into two groups: enzymatic antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, etc) and non-enzymatic antioxidants (ascorbic acid, glutathione, α-tocopherol, carotenoids, flavonoids, melatonin, etc).6 If there are too many oxidants or too few antioxidants, oxidative stress occurs, which causes structural and metabolic alterations in cells; thus, it may cause chronic and permanent damages.6 As in many other diseases such as diabetes mellitus, atherosclerosis, hypertension, cardiac dysrhythmia and myocardial damage, oxidative stress may play a role in the pathophysiology of snake envenomation.7–10
Our aim in this study was to investigate the variations in pretreatment and post-treatment of antioxidant status (TAS), oxidant status (TOS) and oxidative stress index (OSI) levels in snake bites and to learn more about the pathophysiology of snake envenomation. As far as we have determined from the literature, this study is the first clinical study in this area.
Materials and methods
Study population and protocol
The study was conducted at Gaziantep University, School of Medicine, Departments of Emergency and Clinical Biochemistry, between May 2009 and October 2010. The study protocol conformed to the principles of the Helsinki Declaration and was approved by the Gaziantep University Ethics Committee no. 06-2009/257, dated 18 June 2009. All subjects were informed about the study protocol, and written consent was obtained from all participants.
This study was performed without gender discrimination on adult patients over 16 years of age. The study group consisted of 47 consecutive patients who were hospitalised for the treatment of snake envenomation. The control group consisted of 20 healthy individuals who were administrative staff at our hospital.
All participants had normal body habitus. All the participants submitted a detailed medical history and underwent a physical examination by the investigating physicians. Subjects with the possibility of coronary artery disease, hypercholesterolaemia, hypertension, neurological disorders, diabetes mellitus, liver and kidney diseases, peripheral vascular disease, lung disease, iron deficiency, anaemia and obesity found after medical history, physical examinations and laboratory examinations were excluded from the study.
Blood sample collection
Blood samples for the TOS-TAS study were taken from the patients and the healthy control group on admission. Blood samples for laboratory measurements of patients with snake envenomation were taken again after 1 month. Blood samples were withdrawn from a cubital vein into blood tube with lithium heparin and immediately stored on ice at 4°C. The serum was then separated from the cells by centrifugation at 5000 rpm for 10 min. The serum that was collected at the top of tube was taken. The serum samples were stored at −80°C until analysis.
Measurement of TAS-TOS and calculation of OSI
TAS and TOS levels were measured using a colorimetric method that was introduced by Erel.11 The results were expressed as millimolar Trolox equivalent per litre (mmol Trolox equivalent/l) for TAS and micromolar hydrogen peroxide equivalent per litre (µmol H2O2 equivalent/l) for TOS. The ratio of TOS to TAS was accepted as the OSI. For the calculation, the resulting unit of TAS was converted to µmol/l, and the OSI value was calculated according to the following formula:
For statistical evaluation, we used SPSS for Windows V.18.0 (SPSS Inc. Chicago, Illinois, USA). As the data were normally distributed and dependent, groups were compared using a paired-sample t test. As the data were not normally distributed and independent, groups were compared using the Mann-Whitney U test and the Kruskal-Wallis test was performed to evaluate the differences of TAS, TOS and OSI levels during blood-taking periods. The relationship between variables was analysed using the Pearson's correlation test. In all comparisons, p<0.05 was considered statistically significant.
In many regions of Turkey, there are a number of different snake species. The southeast region of Turkey is home to venomous snakes of the Viperidae family, including Vipera ammodytes meridionalis, which is the most dangerous species.12 Every year, approximately 50 000 patients (over 16 years of age) are referred to our emergency department (ED) for diagnosis and treatment. Of 43 021 admissions between May 2009 and October 2010, 67 were due to snake bites. Of these patients (n=67), 47 (63.8% male, 36.2% female) were included in the study. Whereas some of the patients were admitted directly to ED, some were referred from other centres. There were no dry bites (venomous snake bite without envenoming) in our cases. The largest incidence was during the warmest midday hours. The average time between snake bite and patients’ admission to ED was 264±224 (40–580) minutes. All patients were conscious on arrival. While 24 patients (51%) were bitten on the lower extremity, 23 patients (49%) were bitten on the upper extremity. All of the patients developed severe pain, oedema, and subcutaneous bleeding on the extremity where the snakes bit. Also, three patients developed gingival bleeding and two patients developed light conjunctival bleeding (table 1). The mean platelet level was low (146.65±56.22×10³ µl). The mean international normalised ratio level was high (1.41±0.22). All patients were followed in the emergency monitoring room. All of the patients were treated with antivenom (viper venom antiserum, European (equine)) and supportive therapy (intravenous fluid, analgesia and antibiotherapy). None of our patients required intensive care. None needed fasciotomy or extremity amputation. All patients experienced a good recovery when they were discharged.
No statistical difference existed between the average ages of the patient (39±16 years (16–79)) and control (38±15 years (19–68)) groups. In both groups, there was no correlation among age, gender and the levels of TAS, TOS and OSI. The difference in mean TAS, TOS and OSI levels for males and females in both groups was not statistically significant (p>0.05) (table 2).
TAS, TOS and OSI levels in snake envenomation patients upon their arrival to the ED were higher than the healthy control group's levels, and this increase was statistically significant (p=0.001) (table 2) (figure 1).
When TAS, TOS and OSI results were compared according to the blood samples taking periods, the levels were not statistically significant (p>0.05) (table 2).
When TAS, TOS and OSI results were compared according to the location of the bite, the levels were not statistically significant (p>0.05) (table 2).
Comparison of the routine laboratory parameters, coagulation, whole blood and cardiac parameters for the patients with snake envenomation are presented in table 3.
The balance between the oxidant and antioxidant defences in the human body has important health implications. Oxidative stress can occur due to an overproduction of oxidants, overexposure to exogenous oxidants, a decrease in antioxidant defences or a combination of these factors. In these cases, antioxidative mechanisms may remain insufficient to prevent oxidative damage completely. Consequently, oxidative stress, which has been implicated in more than 100 disorders, some of which are present in snake envenomation (such as hypertension, cardiac dysrhythmia and myocardial damage) develops.3 ,6 ,12 ,13
Snake venoms typically contain 30 to more than 100 complex protein toxins.14 Some of these proteins exhibit enzymatic activity, whereas several others are non-enzymatic proteins and polypeptides. Snake venoms exhibit a wide range of biological properties and actions, as well as chemical compositions, toxicity, and pharmacokinetic and pharmacodynamic characteristics.14 The most common snake venom enzymes include acetylcholinesterases, L-amino acid oxidases, serine proteinases, metalloproteinases and phospholipases A2 (PLA2).14 These substances may cause oxidative stress owing to many effects. For example, L-amino acid oxidases, one of the most common components of snake venoms, is a flavoenzyme catalysing the stereospecific oxidative deamination of L-amino acids to give the corresponding a-keto acid.10 ,14 Flavoenzyme oxidase plays a role in the formation of hydrogen peroxide (H2O2). H2O2 is a pro-oxidant/oxidant substance and has apoptotic activity in vascular endothelial cells.10 ,14 Venom PLA2 enzymes share similar structure and catalytic function to mammalian enzymes. However, many venom PLA2 enzymes, in contrast to mammalian enzymes, are toxic and induce a wide spectrum of pharmacological effects.15
Many substances in snake venom affect multiple enzymes and ion channels in the human body. Currently, only a small number of snake venom complexes are fully understood.15 Gaining more in-depth knowledge of the biophysical interactions and structures of snake venom complexes will help us better understand the effects of snake venom, as well as the relationship between oxidants/antioxidants and snake venom.
We have not found any study report in the literature regarding the relationship between snake envenomation and oxidants/antioxidants. To our knowledge, this is the first paper on this topic. The results obtained in this study showed that the oxidative/antioxidative balance shifted toward oxidative status because of snake envenomation. Therefore, we think that oxidative stress plays a role in the pathophysiology of snake envenomation. Evidence of high intakes of antioxidants such as carotenoids, vitamin E, vitamin C and selenium associated with a lower risk for many diseases has been shown in studies involving large groups of men and women.16–18 We think that antioxidant supplementation may have beneficial effects for snake envenomation treatment. Therefore, antioxidant supplementation could be considered as part of the treatment for snake envenomation.
The number of patients in our study was small. We think that the results would be more significant with more patients. Clinically, we did not have very serious cases. Because of this, we did not evaluate the possible difference on TOS/TAS in very serious cases. Also, only TAS and TOS were examined in the present study. Checking subparameters along with TAS and TOS will contribute to a greater understanding of the snake envenomation pathophysiology.
In conclusion, we observed a significant increased in TOS and OSI levels in patients with snake envenomation. This meant that increased oxidative stress was present in patients with snake envenomation. We thought that the snake bite was associated with a shift to an oxidative state. However, these results are not sufficient to explain the relationship between oxidants/antioxidants and snake venom. Further studies are needed to clarify the possible mechanisms underlying the decreased and increased activity and levels. During treatment, antioxidant supplementation may lead to an increase in the antioxidant defence system and thus an improvement in clinical symptoms. Therefore, antioxidant supplementation could be considered as part of the treatment for snake envenomation.
Contributors SZ: study concept and design, acquisition of the data, analysis and interpretation of the data, drafting of the manuscript, statistical expertise. BA: study concept and design, critical revision of the manuscript for important intellectual content. PY: study concept and design, acquisition of the data. RG: acquisition of the data. MO, ST: administrative, technical or material support. CY: critical revision of the manuscript for important intellectual content.
Competing interests None.
Ethics approval The study protocol conformed to the principles of the Helsinki Declaration and was approved by the Gaziantep University Ethics Committee no. 06-2009/257, dated 18 June 2009.
Provenance and peer review Not commissioned; externally peer reviewed.