The effect of sulfur baths on hemorheological properties of blood in patients with osteoarthritis

Balneotherapy is an effective treatment method in various diseases and commonly used treatment modality among patients with musculoskeletal disorders. Sulfur baths are known for healing properties however effect on rheological properties is unstudied. Thus the aim of our study was to determine the effect of sulfur balneotherapy on hemorheological blood indices. A total of 48 patients with osteoarthritis were enrolled to the study. Blood samples were collected twice, before and after 3-week time period. We evaluated complete blood count, fibrinogen, hs-CRP and blood rheology parameters such as elongation index (EI), half-time of total aggregation (T1/2) and aggregation index (AI) analyzed with the Lorrca Maxis. Mean age of studied cohort was 67 ± 5 years. After sulfur baths WBC count was significantly decreased is studied group (p = 0.021) as well as neutrophile count (p = 0.036). Red blood cell EIs were statistically higher after sulfur baths in shear stress ranging from 8.24 to 60.30 Pa. T1/2 was significantly higher (p = 0.031) and AI lower (p = 0.003) compared to baseline. No significant changes in fibrinogen and hs-CRP were observed. It is the first study that evaluate effect of sulfur balneotherapy on rheologic properties of blood. Sulfur water baths may improve erythrocyte deformability and aggregation parameters.

Balneotherapy is commonly used as a part of a treatment modality among patients with musculoskeletal disorders and has been found to be effective in the therapy of various disorders 1,2 . The mechanism of abovementioned therapy is unclear. It was proved that sulfur baths reduces tonicity, pain and joint swelling that partially is caused by increased sodium excretion, which stimulates renal diuresis that improve joints mobility 3,4 . According to Lengwant et al. groups treated with both physiotherapy and sulfur-salt water baths had significantly better outcomes in comparison to groups that were only treated with similar physical therapy, without baths. Balneotherapy procedures were found to have a significant impact on improvement of the joint function, the gait efficiency and kinematics. Moreover, balneotherapy was proved to have a meaningful effect on alleviation of pain and improvement in the quality of life 5,6 .
The most important natural resource of the Solec Zdrój Health Resort (SZHR) is healing sulfide water, considered the most effective in Poland. Due to its composition, the sulfide water in the SZHR is not subject to any chemical amelioration. The ideal parameters of the sulfide water in the spring, classified at the level of 137 mg H 2 S/l, means that the healing sulfide waters in the Solec Zdrój Health Resort are not diluted. Hydrogen sulfide in combination with sulfur, fluorine, iodine, bromine and boron affects the healing properties. The SZHR uses the private "Solec Shaft". The health resort specializes in the treatment of diseases of the musculoskeletal system: degenerative, rheumatic, dermatological and neurological. The main active factor in the healing waters of the SZ is the sulfide ion, which is absorbed through the skin and reaches all tissues of the body through the blood 7,8 . Antioxidant effect H 2 S suppress reactive oxygen species and reactive nitrogen species and increases the expression of antioxidant enzymes 9 . H2S reduced the pro-inflammatory status induced by IL-1β in cultured human cellural chondrocytes. Moreover, H 2 S was found to have a protective effect against the degradation of the matrix in ex vivo experiments in osteoarthritis (OA) cartilage explants 10 . Recent studies in rat models shown that balneotherapy in sulfur-rich water diminishes the presence of oxidative damage markers, cartilage destruction and pain levels thus may be beneficial in non-pharmacological treatment of OA 11 . Karagülle et al. 12 provided preliminary insights into the "biological truth" about natural H 2 S waters and their potential therapeutic role in balneology and health resort medicine.
To our knowledge the effect of sulfur water baths on rheological blood properties was not studied before. Thus the main aim of our study is to determine the effect of sulfide baths during a 3-week stay of patients in the SZHR on hemorheological blood indices, which include complete blood count, fibrinogen and deformability along with aggregation of red blood cells in patients with osteoarthritis.

Materials and methods
Participants. We enrolled 48 subjects (24 females and 24 males). Amongst them 35 participants (17 females and 18 males) underwent regular sulfur baths in Solec Zdrój Health Resort in Poland and 13 subjects (7 females and 6 males) was qualified to the control group without sulfur baths. Study inclusion criteria were: age between 60 and 80 years and diagnosis of osteoarthritis. Exclusion criteria included rheumatologic disorders, smoking, active infection and neoplasms. Participants were fully informed of the study details and expressed their written consent prior to taking part in the study. All the procedures complied with the Declaration of Helsinki and its further amendments 13 . Methods. The intervention group, consisting of 35 subjects, was qualified to fifteen sulfur baths in temperature 34-37 °C, each lasting 15 min. Sulfur baths were performed weekdays during 3-week stay in the Solec Zdrój Health Resort (Poland). Both study groups underwent similar standardized physical therapy sets consisting of kinesiotherapy, massage, manual therapy and laserotherapy. All interventions were supervised by medical doctor and nurse. Blood samples from all patients were collected at the beginning of treatment stay in the SZHR (baseline) and after a 3-week period (21 days). Qualified nurse collected twice 10 ml of fasting blood from the cubital vein into Vacuette EDTA K2 vacuum tubes. Hemorheological blood indices were determined in the Blood Physiology Laboratory of the University of Physical Education in Krakow and in the Diagnostyka S.A. laboratory in Krakow (Poland).
No statistically significant differences between studied groups at baseline measurements were found. We observed statistically significant differences between WBC, HGB, MCH, MPV, NEU parameters after sulfur baths. Detailed complete blood count results are presented in Table 2.
We have not found statistically significant differences between analyzed groups in high sensivity C-reactive protein concentrations, fibrinogen levels and coagulation parameters as shown in Table 3.
Assessment of red blood cells aggregation parameters in intervention group revealed statistically significant changes in half-time of total aggregation and aggregation index as presented in Table 4.
Assessment of red blood cells deformability parameters in intervention group revealed statistically significant changes in Red blood cell EIs. Were statistically higher after sulfur baths in shear stress ranging from 8.24 to 60.30 Pa as shown in Fig. 1.

Discussion
According to our knowledge, there are no clinical reports that address the effect of balneological therapies, especially sulfur water baths, on combined rheological and biochemical blood parameters in a comprehensive and standardized study.
We found that rheological parameters described by elongation index, which determine red blood cell length changes in relation to width while stretched, were improved after Solec Zdrój Health Resort treatment. Those results were significantly higher while using increased shear stresses such as 8.24, 15.98, 31.03 and 60.30 Pa. The deformability of red blood cells has an important role in their main function, which is the transportation of  Table 3. Mean values (± SD-standard deviation) or median (interquartile range) of the selected biochemical and blood coagulation parameters among health resort patients before and after treatment stay and in the control group. *Significant difference (p < 0.05).  Table 4. Mean values (± SD-standard deviation) of red blood cells rheological parameters among health resort patients before and after treatment stay and in the control group. *Significant difference (p < 0.05).  [19][20][21] . Additionally, Gelmini et al. 22 found that red blood cells deformability lowers with age which affects tissue oxygenation. Franzini et al. 23 found that significant decrease in the deformability of red blood cells among older patients was connected with increased membrane cholesterol which affects membrane viscosity. The exact physiological mechanism that the sulfur bath induces on the hemorheological parameters is not known. H 2 S shows antioxidant properties, as it quenches reactive oxygen species (ROS) and reactive nitrogen species (RNS) and increases the expression of antioxidant enzymes by activating the transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf-2) 9 . It is known that ROS cause vascular cell damage, the recruitment of inflammatory cells, lipid peroxidation, collectively leading to vascular remodeling 24 . Taking abovementioned into consideration it could be hypothesized that anti-oxidant properties of sulfur baths may improve hemorheological blood properties. Moreover, hydrogen sulphide forms fat-soluble polysulphides in the skin, that allows to enter the bloodstream through the capillaries. It is hypothesized that sulfur baths positive effect on the rheological properties of blood might be connected with endothelial cells, so they can regulate nitric oxide (NO) discharge, which is strong vasodilatory and anti-inflammatory signaling molecule 25 . NO synthesis in endothelial cells is controlled by many factors, including the shear forces acting on the vessel walls, which in turn are determined by the flow and viscosity of the blood in the peripheral part of the vessel 26,27 . In small-diameter capillaries, erythrocytes flow in one row, using the maximum capacity of their deformability. Increased blood flow in the small arteries of fingers was reported in patients with rheumatoid arthritis after bathing in sulphide water 5 . This is in line with our study where we found increased erythrocytes deformability in shear stresses including 8.24, 15.98, 31.03 and 60.30 Pa. It is important not only to evaluate the red blood cells deformability index but also RBC aggregation parameters. Interestingly we found that the time required for half maximal change in aggregation signal (T 1/2 ) of RBC was significantly lower among patients treated in health resort with sulfur water baths. It was comparable with previous study among triathlon subjects 22 . Factors affecting the formation of red blood cells aggregates may be divided into two groups. The first group consists of external factors, includes the level of plasma proteins (fibrinogen, lipo-proteins, macroglobulins, immunoglobulins), shear forces and hematocrit. The second group consists of internal factors, such as the shape of erythrocytes, their deformability and the properties of the cell membrane 28 . Moreover, patients treated in health resort had significantly lower results of the extent of red blood cells aggregation described with aggregation index (AI). Similarly to deformability, the aggregation of red blood cells has an important impact on their main function-transportation, however the lower the aggregation index the better and the higher T 1/2 the better as well. We did not find any significant differences in red blood cells count between study groups, similar results were found by Galvez 29 . According to Baskurt et al. rheological properties of blood mainly results from the properties of red blood cells, which strongly modify blood flow in blood vessels, but also from the properties of plasma, including proteins contained in it. The presence of high molecular weight proteins, such as fibrinogen, lipoproteins and globulins (especially α2-macroglobulin and immunoglobulins), increase plasma viscosity and thus increase blood viscosity 30 . We found that the level of white blood cells (WBC) significantly lowered among patients with osteoarthritis treated in SZ health resort. Speaking of granulocytes, which are the types of WBC, the biggest difference was seen in neutrophiles, which number has also significantly decreased. Such results may suggest that treatment in sulfur water in health resort has positive impact on chronic low-grade inflammation process as presented by Xu et al. 31 However, we did not observe any significant differences in high sensitive C-reactive protein concentrations. Our results are in opposition to those presented by Olah, in which CRP significantly decreased, nevertheless, www.nature.com/scientificreports/ second blood specimen collection time was different between studies, 3 weeks versus 3 months accordingly 25,26 . In some cases, the rheological properties of blood depend on fibrinogen. Its lower level affects the initiation of red blood cell aggregation, which leads to increased blood viscosity. Fibrinogen is a 340 kDa glycoprotein synthesized in the liver, with plasma concentrations of approximately 150-400 mg/dl; is a protein involved in blood coagulation and hemostasis, it is also involved in inflammation and tissue repair processes. Fibrinogen facilitates platelet aggregation by binding the glycoprotein IIb/IIIa receptor and forming a fibrin monomer that rapidly polymerizes to form a clot 32,33 . According to Sen et al. plasma fibrinogen levels increase 2-to 3-times during the inflammatory response, which causes cell aggregation and increases blood viscosity 34 . The rheological properties of blood depend to a large extent on the fibrinogen concentration. Its elevated level leads to the increased red blood cell aggregation, which result in increased blood viscosity. However, we have not found statistically significant differences between analyzed groups in fibrinogen levels and coagulation parameters.
Moreover, we found that the mean hemoglobin levels among patients treated in health resort significantly decreased after 3 weeks. It may be relative, due to increased thirstiness after bathing in sulfide waters that induces overhydration. Abovementioned situation results in fluctuation of blood pressure and depending on the speed of this reaction and on the adaptive capacity of the human body, the heart rate may also alter 35 . Limitations. The study has limitations. Firstly, the control group was smaller than intervention one however there were no statistically significant differences between these groups in sex ratio, age and other baseline parameters. Furthermore, study design was prospective. Secondly, we have performed measurement at baseline and after 3-week time that reflects course of sulfur balneotherapy. Average RBC lifespan in normal individual is 115 days 36 . Thus, when taking it into account, effects on red blood cell hemorheological properties are probably even underestimated. Thirdly, along with the laboratory parameters functional assessment with quality of life evaluation could be performed, but our study intended to assess biochemical and rheological background of sulfur balneotherapy.

Conclusions
Osteoarthritis is a widespread disease causing pain and inflammation that limits mobility and functionality 37,38 . Despite high prevalence of osteoarthritis, the effective treatment is undetermined. Sulfur water baths may improve erythrocyte deformability and aggregation parameters in patients with osteoarthritis along with reducing of neutrophile levels. The efficacy of balneotherapy, especially sulfur water baths should be the subject of further research.

Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.