Original article 

Giuseppe Bonforte, Simona Zerbi 1, Stefania Longoni 2, Marianno Franzini 3 

Nephrology and Dialysis Unit, S. Anna Hospital, Como; 1 Nephrology and Dialysis Unit, Seriate Hospital, Bergamo; 2 Nephrology and Dialysis Unit, Desio Hospital, Milano; 3 Ozonotherapy Teacher, Università degli Studi di Pavia

Running title: Ozonotherapy and dialysis

Correspondence:  Bonforte Giuseppe MD

Ospedale “Sant’Anna” 

Via Napoleona n 6, 22102  Como

Abstract 

Peripheral arterial disease and secondary amyloidosis account for significant morbidity and mortality among uremic patients. Several papers demonstrated the beneficial effect of ozonotherapy in the treatment of vascular and orthopedic diseases. We performed a study regarding its impact on these severe complications in hemodialysis patients.

Twenty-five hemodialysis patients were studied. Ten patients suffered from stage III – IV peripheral arterial occlusive disease according to Fontain (five of them had previous unilateral lower limb amputation) (Group 1). Fifteen subjects had remarkable joint pain due to deposition of  b2-microglobulin (Group 2). All patients were treated by analgesic therapy. SIOOT protocols were applied: patients received major O₃ autohemotherapy, sub cutis ozone infusion and body exposure to oxygen/ozone mixture by bagging. A questionnaire was submitted at the beginning and at the end of ozonotherapy for the individual assessment of the efficacy of the treatment.  

Complete healing of  ischemic ulcers was obtained in 7 patients. 88% of patients reported a subjective successful outcome after ozonotherapy. Both Group 1 and Group 2 subjects reported a significant decrease in pain perception. The use of analgesic drugs was reduced in 84%. 75% got a normal sleep/wake cycle. An enhancement in physical activity was reported by 64% of subjects. 

Ozonotherapy improves hemodialysis patients’ quality of life reducing bone pain perception and attenuating clinical signs of peripheral vascular disease. 

Key words: ozone therapy, dialysis, peripheral vascular disease, lower limb ischemia, gangrene, b2-microglobulin, secondary amyloidosis

Ozonotherapy is known from more than fifty years. Several papers confirmed the beneficial effect of oxygen-ozone therapy in the treatment of vascular disorders (1-3), osteoarthritis (4,5), orthopedic pathology (6) and various pain syndromes (4,5). Ozonotherapy is commonly performed by minor O₃ autohemotherapy and major O₃ autohemotherapy (7). The precise control of ozone dosage is mandatory in order to avoid serious toxic side-effects.

Patients with end-stage renal disease attending dialysis are particularly exposed to the development of peripheral arterial occlusive disease (8) and secondary amyloidosis (9). Lower limb amputation (10), carpal tunnel syndrome, remarkable pain in the shoulders, cervical and lumbar column are common in subjects with long time from initiation of dialysis. The optimal management of these complications in end-stage renal disease patients is quite controversial. Thus we performed a prospective study regarding the impact of ozonotherapy on peripheral artery disease and pain in uremic patients on maintenance hemodialysis.

PATIENTS AND METHODS

Twenty-five uremic patients on maintenance hemodialysis were studied (12 female, 13 male), aged 70 years (range 48-97) with end stage renal disease treated with hemodialysis for 10 years (range 4-34). 

Ten patients suffered from stage III-IV peripheral arterial occlusive disease according to Fontain  (five of them had previous unilateral lower limb amputation) (Group 1). Fifteen subjects had remarkable joint pain due to deposition of  b2-microglobulin (Group 2). In group 1 renal diseases included 4 patients with diabetic nephropathy and 6 patients with ischemic nephropathy. In group 2 renal diseases consisted of chronic pyelonephritis (n=8), ischemic nephropathy (n=2), chronic pyelonephritis (n=5). 

All patients underwent regular three times per week bicarbonate hemodialysis treatment and were treated by analgesic therapy. The hemodialysis adequacy (11) and the levels of hemoglobin were determined monthly.

Ozone generator (MULTIOSSIGEN Medical 95 CPS, Gorle, Bergamo, Italy) was used in the study. SIOOT protocols (12) were applied. Group 1 received major O₃ autohemotherapy (250 ml of blood or physiologic solution placed in contact with 150 cc of an oxygen/ozone mixture with a final concentration of 50 mg/ml), ischemic leg exposure to ozone by bagging (oxygen/ozone mixture at a concentration of 3–30 mg/ml per 10 min) and sub cutis ozone infusion  around ischemic ulcers (50 cc of oxygen/ozone mixture at a concentration of 3 mg/ml); procedures were performed twice a week for at least 8 weeks. Group 2 received major O₃ autohemotherapy (250 ml of blood or physiologic solution  placed in contact with 150 cc of an oxygen/ozone mixture with a final concentration of 50 mg/ml) and sub cutis ozone infusion in painful areas such as shoulder, knee, wrist and/or vertebral column (25-50 cc of oxygen/ozone mixture at a concentration of 2 to 15 mg/ml); procedures were performed twice a week for at least 1 month and then every seven or fifteen days according to the patient’s response. 

A questionnaire was submitted at the beginning and at the end of ozonotherapy for the individual assessment of the efficacy of the treatment: pain assessment by a 0-10 point Visual Analog Scale (VAS), exercise ability and analgesic drugs consumption were evaluated. 

The changes of continuous variables were assessed by ANOVA for repeated measurements. A p value < 0.05 was considered significant. Statistical analysis was performed using Statview software (5.0 for Windows). 

RESULTS

All patients completed the protocol. No side effect was observed. Hemodialysis adequacy and hemoglobin levels remained stable during the study. 

In individual assessment of treatment efficacy, 88% reported a successful outcome after ozonotherapy: 14 patients experienced optimal improvement (n=8 secondary amyloidosis; n=2 III stage peripheral ischemic disease; n=4 IV stage peripheral ischemic disease), n=8 reported valid improvement (n=5 secondary amyloidosis; n=2 III stage peripheral ischemic disease; n=1 IV stage peripheral ischemic disease), n=3 observed no changes relative to baseline (n=2 secondary amyloidosis; n=1 IV stage peripheral ischemic disease) (Tab. 1).  

Both Group 1 and group 2 subjects reported a similar and significant decrease in pain perception (Fig. 1 and Fig. 2 respectively) (p<0.001; F=164.89). At the same time the use of analgesic drugs was modified: 4 patients required dosage reduction, 21 stopped consumption. 75% got a normal sleep/wake cycle. 

An enhancement in exercise activity was reported by 64% of subjects. An improvement in walking ability was observed in patients suffering from peripheral arterial occlusive disease without previous unilateral lower limb amputation. Ischemic limb ulcers improved in 8 patients: a complete healing of ischemic ulcers was obtained in 7 patients. 

Beneficial effects lasted for 2 months. 

DISCUSSION

The over sixty-five years age group is the emergent segment of the population presenting for dialysis. The prevalence of the elderly dialysis group is also high. Both age and chronic renal failure are risk factors for peripheral vascular disease and joint pain (due to arthritis and b2-microglobulin deposition respectively). Vascular nephropathies represent the major cause of end-stage renal disease, followed by diabetes. The death causes are chiefly cardiac related and the main prognostic factors are rate and severity of comorbidities. Vascular disease and amyloidotic arthropathy represent two of the more critical aspects of dialysis in the elderly (13,14) and contribute to strongly impair patients quality of life by reducing walking and exercise ability. 

Peripheral arterial occlusive disease accounts for significant morbidity and mortality among end-stage renal disease patients. Its prevalence appears to be much higher among uremic patients than in the general population. Hazards probably include both conventional and dialysis or uremia-associated risk factors. Despite the fact that this is a common disease in uremic patients, most of them are not screened for peripheral arterial disease; moreover prevention by smoking cessation, preventive foot care and exercise are not systematically applied to these subjects (8). 

Amputation is more common in hemodialysis patients than in the general population. Male sex, diabetes, previous diagnosis of peripheral vascular disease, mean systolic blood pressure and elevated serum phosphorous level are associated with the outcome of amputation; among patients without diabetes, a previous diagnosis of cardiac disease, longer time from initiation of dialysis therapy and previous hospitalization for limb ischemia are associated with increased risk for future amputation (10). Thus the importance of preventing amputation in this population cannot be overemphasized and nephrologists should be aware of the remarkable value of detecting peripheral vascular disease by screening every dialysis patient. The optimal management of ischemic ulceration and gangrene in end-stage renal disease patients is quite controversial. The management of peripheral arterial occlusive disease includes drug therapies, limb-sparing procedures such as percutaneous angioplasty and vascular reconstruction. Medications have little verified benefit (15,16). Diffuse distal lesions and vascular calcification in dialysis patients often impair angioplasty successful; moreover serious comorbid conditions make hemodialysis patients poor candidates for surgical procedures and contribute to increase mortality rate for those who undergo limb-sparing procedures (8). Hence non-invasive therapeutic procedures are desirable for this class of patients. 

Dialysis-related amyloidosis secondary to b2-microglobulin deposits is a common complication of dialysis. It preferentially locates in the osteoarticular tissues, particularly in large bones close to joint spaces, and synovial membranes although small deposits are also found in various organs, mainly the heart and gastrointestinal tract. Pathologic studies have demonstrated a high prevalence of articular b2-microglobulin early in the course of hemodialysis and peritoneal dialysis, antedating clinical manifestations by several years and positively correlating to time from initiation of dialysis therapy. The key factor in the pathogenesis is retention of b2-microglobulin associated with secondary modifications of the molecule such as limited proteolysis, conformational changes and the formation of advanced glycation end products (17). Biocompatible membranes for dialysis remove and adsorb b2-microglobulin more efficiently than the cellulosic membranes, but they are not enough to erase the disease. Clinical manifestations are likely associated with the inflammation observed when the deposits involves capsules and sinovia with recruitment of macrophages around the deposits (9). Patients develop carpal tunnel syndrome, remarkable pain in the shoulders, cervical and lumbar column. The management of secondary amyloidosis mainly consists of analgesia, leading to a high risk of gastro enteric bleeding. 

Several papers confirmed the beneficial effect of oxygen-ozone therapy in the treatment of vascular disorders (1,2,3), osteoarthritis (4,5), orthopedic pathology (6) and various pain syndromes (4,5). As peripheral arterial disease and secondary amyloidosis account for significant morbidity among end-stage renal disease patients, we performed a prospective study regarding the impact of ozonotherapy on peripheral artery disease and bone pain in hemodialysis patients. 

Ozone therapy is commonly performed by minor O₃ autohemotherapy and major O₃ autohemotherapy (7); other medical uses include extracorporeal circulation, rectal insufflation and almost-total body exposure (5). Topical applications include ozonated oil (18) and water (5), and intrarticular applications (6). Ozone is known to be a strong oxidant. When dissolved in the blood, it produces a number of reactive oxygen species (ROS) that are almost completely quenched by antioxidant systems present in plasma and blood cells (19) at safe range of ozone concentration. Some ROS react with polyunsaturated fatty acids (PUFA) and generate hydrogen peroxide and lipid oxidation products; the latter is an important messenger responsible for transmitting beneficial effects of ozone. Ozone effects includes glycolysis activation and ATP levels enhance, a slight induction in cytokins production, increased NO production (5); the increase in 2,3 -diphosphoglycerate levels (responsible for enhanced oxygen delivery to ischemic tissues) is still controversial (20). An increase in antioxidant enzymes was observed, probably carrying a tolerance to chronic oxidative stress (5). 

The beneficial effect of ozonotherapy on peripheral ischemic disease may be due to a group of mechanisms that improve blood flow in hypoxic areas and decrease the symptoms of ischemia: decrease in blood viscosity and coagulation (20,21), vasorelaxation (22) and alterations in prostanoid production (23). Regarding osteoarticular pain, sub cutis ozone infusion in muscle trigger points induces noxious inhibitory control and subsequent muscle relax, pain reduction and vasodilatation (5). 

The therapeutic ozone dose ranges from 20 to 80 mg/ml per gram of blood. A problem concerning the application of ozone in medicine is its induction of oxidative stress. Hemodialysis patients are known to be particularly exposed to generation and deleterious effects of free radicals, due to uremia and dialysis procedures (24). Oxidative stress is involved in several disease complications such as secondary amyloidosis and atherosclerosis (25,26). As a result an objection may be that ozone extends oxidative stress even at low doses. Ozonotherapy’s influence on oxidative stress in hemodialysis patients has already been studied. Tylicki et al. demonstrated that ozonated autohemotherapy with ozone concentration 50 mcg/ml per gram of blood, applied three times a week, induces no oxidative cell injury in the hemodialysis population: the antioxidant defence system neutralizes oxidative properties of ozone at this concentration and protects against oxidative cell damage (2). Therefore ozonotherapy is a safe method and may be a complementary clinical approach in hemodialysis patients when correct therapeutic doses are applied. 

In the present paper subjective clinical improvement was reported by the majority of patients. A placebo effect, at least in part, may have played a role in limiting this measure based on subjective information; nevertheless clinical observations and the absence of changes in factors able to affect patients’ outcome (hemodialysis adequacy, anemia) eliminate such a theory. Complete healing of ischemic ulcers was obtained in a high percent of patients. Individual decrease in pain perception was strengthened by analgesic dosage reduction or suspension. 

Bone pain perception and clinical signs of peripheral vascular disease reduction were observed. Subsequent achievement of normal sleep/wake cycle and better physical activity improved patients’ quality of life. The concomitant reduction in analgesics use reduced the risk of gastro enteric bleeding, a frequent complication in dialysis patients. 

The result was obtained by a non-invasive therapeutic procedure; as serious comorbid conditions make hemodialysis patients poor candidates for surgical procedures, ozonotherapy may become a desirable therapeutic option for this class of patients. 

The present study, although uncontrolled, demonstrates the beneficial clinical effect of ozonotherapy in patients on maintenance hemodialysis. Anyway given the small sample size and design limitations, large-scale clinical trials should be done to give evidence to clinical practice and to attest the long-term beneficial effects of this therapy. 

TABLES

Tab. 1

Figures

Fig. 1 Pain perception before and after ozonotherapy in secondary amyloidosys.

Fig. 2. Pain perception before and after ozonotherapy in secondary amyloidosys. 

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