Investigation of oxidant-antioxidant status in patients treated with hirudotherapy: an experimental study

Objective: To examine the influence of hirudotherapy on parameters of oxidative stress. Method: The cross-sectional study was conducted from March 29 to September 29, 2021, at the Alanya Research and Training Hospital’s Traditional and Complementary Medicine Application Centre, Turkey, and comprised adult volunteers of either gender. The participants were subjected to two sessions of hirudotherapy 4 weeks apart. Total antioxidant status, total oxidant status, oxidative stress index values, ischaemia-modified albumin level, paraoxonase 1, disulfide, native thiol, total thiol, and arylesterase levels were assessed at baseline and after the second hirudotherapy session. Data was analysed using SPSS 15. Results: Of the 50 subjects, 30(60%) were females and 20(40%) were males. The overall mean age was 47.10±15.16 years. Oxidative stress, ischaemia-modified albumin and disulfide levels decreased, but not significantly (p>0.05). The reduction in disulfide levels was significant (p=0.021). Conclusion: Hirudotherapy, within its limitations, could reduce oxidative stress.


Introduction
Hirudotherapy (HT) is the process of the application of medicinal leeches like Hirudo Medicinalis and Hirudo Verbana to the patient by certified physicians.These leaches are obtained from leech breeding businesses. 1 Leeches are parasitic organisms that feed on blood and have more than 650 species.They live as temporary ectoparasites in mammals and other vertebrates, such as fish and frogs. 2 Leeches have been widely used since ancient times to lower blood pressure.Leeches provide benefits both by sucking blood from the host, and with the bioactive substances they give to the host while they are sucking blood.There are various bioactive substances detected in the saliva of medicinal leeches.Some of these substances are hirudin, tryptase inhibitors and histaminelike substances, eglin, bdellin, hyaluronidase, apyrase, calin, destabilase, hirustatin, acetylcholine, complement inhibitors, and carboxypeptidase-A inhibitors.Such bioactive agents have coagulation inhibition, antiaggregant, fibrinolytic, antimicrobial, analgesic, and antiinflammatory effects 3 (Table 1).
The World Health Organisation (WHO) describes traditional medicine as the practice of preventing, treating or diagnosing physical or psychological disorders, depending on beliefs and experiences unique for various cultural characteristics.Between 2014 and 2023, WHO supported member states in developing policies that will strengthen the role traditional medicine plays in keeping people healthy. 4In 2004, the United States Food and Drug Administration (FDA) permitted the trade of medicinal leeches in the US and their usage for general purposes, in plastic surgery, and in microsurgery applications.Physicians had refrained from using traditional and complementary medicine until recently, but nowadays, when its benefits have been shown by scientific research, they have turned to this field again.
Oxidative stress (OS) is defined as a phenomenon resulting out of a discrepancy between the production of free oxygen radicals in cells and tissues, and the capacity of the organism to detoxify these reactive species. 5 refers to a development that emerges during usual metabolic activities.When OS increases, cell, tissue and organ damage can occur.Therefore, an increase in OS should be prevented or balanced with antioxidants.There are many structures that take a role in the antioxidant system and neutralise oxidant substances.Increased OS can lead to undesirable consequences, such as premature aging, atherosclerosis, cancer, Parkinson's disease and Alzheimer's disease. 5,6veral techniques have been proposed to determine OS and antioxidant status.The current study was planned to examine the influence of HT on new-generation oxidant and antioxidant parameters.

Materials and Methods
The cross-sectional study was conducted from March 29 to September 29, 2021, at the Alanya Research and Training Hospital's Traditional and Complementary Medicine Application Centre, Turkey, and comprised adult volunteers of either gender.
It has been recommended to include at least 30 participants in studies involving parametric tests, the current study inflated the sample size to account for potential data losses. 7The subjects included furnished written informed consent.The exclusion criteria were: bleeding diathesis, such as haemophilia, severe anaemia with haemoglobin (Hb) <9.5g/dl), use of anticoagulant drugs, such as warfarin, pregnancy and breastfeeding, history of allergies to leeches, chronic kidney failure and dialysis, presence of pacemaker, age <18 years, chemotherapy, active bleeding, like haemorrhoids, normal menstrual cycle, postoperative period, etc., and glycated Hb (HbA1c) >10, or fasting blood sugar (FBS) >250mg/dl.Approval was received from Alanya Alaaddin Keykubat University Clinical Research Ethics Committee.
After measuring height, weight and blood pressure of the participants and taking down their blood analyses, they were subjected to 2 HT sessions with an interval of 4 weeks.A total of seven medium-sized leeches weiging approximately 1-1.5g were used per person for HT in each session.The leeches were placed in the lumbosacral region.They were placed on the surface of the skin and separated spontaneously within 1-3 hours.The leeches that separated from the skin were considered medical waste and were thrown into a red medical waste bag.The places where the leeches were separated were cleaned with polyvinylpyrrolidone antiseptic solution and covered with hard cotton.At the end of the second HT session, the baseline blood tests were repeated.
Venous blood samples were taken from the participants for FBG, HbA1C, international normalised ratio (INR), and haemogram.OS status was determined.Around 5cc of blood was collected in a biochemistry tube with ethylene diaminetetraacetic acid (EDTA) and centrifuged for 10 minutes at 1500rpm, and the serum part was taken for analyses.The serum portion was transferred to Eppendorf tubes using Pasteur pipettes.The Eppendorf tubes were divided into 2 groups.The blood taken before HT was labelled as Pre-HT, and the blood taken after the second session of HT was labelled as Post-HT.The samples were kept at -80°C (Thermo Scientific Forma 88000, US) until the day of analysis in the Biochemistry Laboratory of Alkü Medical Faculty.
Commercial kits were utilised to measure TAS values (REL assay Diagnostics, Turkey).The automated method was based on the disappearance of the characteristic colour of the relatively stable 2,2' Azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) cation by antioxidants.The assay provides excellent tender results <3% which are presented in units of mmol Trolox equivalent/L. 8mmercial kits were utilised to measure TOS values (REL assay Diagnostics, Turkey).The results are presented in units of micromolar hydrogen peroxide equivalent per liter (μmol H2O2 equivalent/L). 7The ratio of TOS to TAS was used to calculate OSI values.For sums, the resulting unit of TAS was converted to μmol/L, and the OSI value was figured out based on the equation OSI=TOS/TAS. 8N1 and ARES activities of the sera were identified with a fully automated technique (REL Assay Diagnostics; Mega Tıp, Gaziantep, Turkey).Phenylacetate was utilised as a substrate to estimate ARES activity.As a result of the hydrolysis of phenylacetate, phenol and acetic acid were produced.The phenol joined 4-aminoantipyrine and potassium ferricyanide, and was measured using the colorimetric method.The results are presented as units per liter, which is equal to the hydrolysis of 1 micromole of phenylacetate in 1 litre and 1 minute. 8PON1 activity results are presented as units per litre, which are equal to the hydrolysis of 1 micromole of the substrate in 1 litre and 1 minute. 8e albumin cobalt binding analysis method was employed to determine the presence of IMA.The absorbance of the samples was determined at 470nm with a spectrophotometer.The outcomes are presented as absorbance units (ABSUs). 8iol/disulfide homeostasis (TDH), which is the sum of thiol-native and thiol/disulfide variations, was measured using a method developed by Erel and Neselioglu 9 .Half of the difference between total thiol and thiol-native amounts provided the quantity of disulfide bonds. 9TDH analyses were conducted using the automated spectrophotometric technique, with half of the difference between total thiol and thiol-native giving the quantity of the dynamic disulfide. 9ta was analysed using SPSS 15.Baseline and postintervention differences were compared using the parametric independent-samples t-test.Data was expressed as frequencies and percentages as well as mean ± standard deviation, as appropriate.P<0.05 was considered statistically significant.
Biochemical tests showed that there were non-significant differences between baseline and post-intrevention values for TAS, TOS, OSI, PON1, ARES and IMA (p>0.05).The reduction in disulfide levels was significant (p=0.021)(Table 3).

Discussion
In the current study, there were 52% participants who were overweight, and 32% were obese.Obesity is a chronic and treatable disease defined as "excessive fat accumulation in the body due to the energy taken with food being more than the energy consumed".According to the WHO data in 2016, there were 39% adults worldwide who were overweight, and 13% were in the obese category. 10The prevalence of obesity is 39.6% in the US, 11 30-70% in Europe, and 30.2% in Turkey. 12e current study limited itself to 2 sessions 4 weeks apart so that the participants may feel encouraged to complete the study.The waist region (any area between L1 and S2) was chosen for ease of application.OSI, IMA and disulfide levels, which indicate OS, decreased numerically after HT, but only the reduction in disulfide levels was significant (p=0.021).
Despite the finding that reduction in OSI and IMA results was not at the level of statistical significance, the fact that they accompanied a significant decrease in disulfide levels clearly showed that the application of medicinal leeches reduced OS.No relevant study could be found in the current literature investigating the antioxidant effects of leeches.However, in a study by Gröbe A et al., it was reported that leech therapy improved venous obstruction and saved ischemic flaps in 148 patients followed in the plastic surgery service. 13sides, there are studies showing that HT is effective in reducing joint stiffness and pain associated with osteoarthritis. 14Venous blood collection (phlebotomy) has also been shown to reduce OS. 15,16gil et al. 17 reported that wet cupping therapy removed oxidants and reduced OS.Another study found that high iron stores in the body increase OS in neurons.Therefore, these levels were found to increase the risk of Alzheimer's disease.It was predicted that phlebotomy performed without causing anaemia can improve brain functions, slow down neurodegeneration, and improve cognitive and behavioural functions in Alzheimer's disease. 18e OSI is found elevated in many chronic diseases.For example, a direct relationship was determined between OSI and low-density lipoprotein (LDL) and triglyceride (TG) levels in the serum.However, high-density lipoprotein (HDL) levels were not part of such a relationship. 19TOS and OSI were determined as significantly higher among patients with schizophrenia in comparison to those without schizophrenia. 20Therefore, reducing the OSI levels is important.
It is not clear whether the OS-reducing effect noted in the current study was due to the blood sucking of leeches, or due to the bioactive substances in their saliva. 3It could be both, and there is a need for further research on the topic.There were limitations to the current study as it did not include a control group, and no dietary restriction was encouraged between the two sessions.For instance, the intake of foods known to have antioxidant effects was not restricted.

Conclusion
HT, with its limitations, could be used as a method to reduce OS in the treatment of chronic diseases, or in the maintenance of one's current health.