Data Recorded in Real Life Support the Safety of Nattokinase in Patients with Vascular Diseases.

Nattokinase (NK) is a serine protease enzyme with fibrinolytic activity. Even if it could be used for the treatment of several diseases, no data have been published supporting its use patients who underwent vascular surgery. In this study, we evaluated both the efficacy and the safety of nattokinase (100 mg/day per os) in patients admitted to vascular surgery. Patients were of both sexes, >18 years of age, with vascular diseases (i.e., deep vein thrombosis, superficial vein thrombosis, venous insufficiency), and naïve to specific pharmacological treatments (anticoagulants or anti-platelets). Patients were divided into three groups. Group 1: patients with deep vein thrombosis, treated with fondaparinux plus nattokinase. Group 2: patients with phlebitis, treated with enoxaparin plus nattokinase. Group 3: patients with venous insufficiency after classical surgery, treated with nattokinase one day later. During the study, we enrolled 153 patients (age 22-92 years), 92 females (60.1%) and 61 males (39.9%;), and documented that nattokinase was able to improve the clinical symptoms (p < 0.01) without the development of adverse drug reactions or drug interactions. Among the enrolled patients, during follow-up, we did not record new cases of vascular diseases. Attention to patients' clinical evolution, monitoring of the INR, and timely and frequent adjustment of dosages represent the cornerstones of the safety of care for patients administered fibrinolytic drugs as a single treatment or in pharmacological combination. Therefore, we can conclude that the use of nattokinase represents an efficient and safe treatment able to both prevent and treat patients with vascular diseases.

Nattokinase-heparin exhibits beneficial efficacy and safety-an optimal strategy for CKD patients on hemodialysis.

Heparin is a widely used anticoagulant in hemodialysis (HD) for patients with chronic kidney disease (CKD); however, it entails the risk of thrombus formation due to heparin-induced thrombocytopenia. Indeed, CKD patients on HD are associated with excessive mortality from cardiovascular disease due to their prothrombotic profile. Therefore, it would be a significant breakthrough to develop a thrombolytic adjuvant that facilitates heparin to achieve its proper anticoagulant efficiency at a much lower dose for greater safety. Nattokinase (NK), a valuable dietary supplement possessing strong fibrinolytic and thrombolytic activity, was reported to interact with heparin and thereby the beneficial efficacy of NK-heparin was investigated herein. NK-heparin induced a synergistic enhancement of clotting time both in vitro and in vivo evaluations, whereas the overall fibrinolytic activity was only marginally enhanced. Moreover, it was demonstrated for the first time that NK induced potent degradation of all three chains of fibrinogen. In particular, NK-heparin markedly reinforced the fibrinolysis activity of NK, which may underlie, at least in part, the mechanism by which NK-heparin benefited their overall thrombolytic and anticoagulant activity. Collectively, we clarified the beneficial combination efficacy of NK and heparin for greater safety, providing a powerful impetus for physicians to administer heparin to a larger portion of patients with CKD.

Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease.

Natto, a fermented soybean product, has been consumed as a traditional food in Japan for thousands of years. Nattokinase (NK), a potent blood-clot dissolving protein used for the treatment of cardiovascular diseases, is produced by the bacterium Bacillus subtilis during the fermentation of soybeans to produce Natto. NK has been extensively studied in Japan, Korea, and China. Recently, the fibrinolytic (anti-clotting) capacity of NK has been recognized by Western medicine. The National Science Foundation in the United States has investigated and evaluated the safety of NK. NK is currently undergoing a clinical trial study (Phase II) in the USA for atherothrombotic prevention. Multiple NK genes have been cloned, characterized, and produced in various expression system studies. Recombinant technology represents a promising approach for the production of NK with high purity for its use in antithrombotic applications. This review covers the history, benefit, safety, and production of NK. Opportunities for utilizing plant systems for the large-scale production of NK, or for the production of edible plants that can be used to provide oral delivery of NK without extraction and purification are also discussed.

Effects of Escherichia coli subtilase cytotoxin and Shiga toxin 2 on primary cultures of human renal tubular epithelial cells.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause post-diarrhea Hemolytic Uremic Syndrome (HUS), which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB) that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC) and compare its effects with those produced by Shiga toxin type 2 (Stx2), in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubAA272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2.

Combined nattokinase with red yeast rice but not nattokinase alone has potent effects on blood lipids in human subjects with hyperlipidemia.

The purpose of this randomized, double-blind, placebo-controlled, parallel comparison study was to evaluate the lipid-lowering effect of orally administrated nattokinase and nattokinase combined with red yeast rice (RYR) extract on blood lipids in patients with hyperlipidemia. A total of 47 patients with hyperlipidemia were assigned to one of three groups: 1. nattokinase-mono formula (50 mg/capsule), 2. combined formula of nattokinase with RYR (300 mg of extract/capsule) and 3. placebo. Subjects received a twice daily dose of two capsules for six months. The mono formula showed no effects on blood lipids until month six, while the combined formula ameliorated all of measured lipids starting from month one. In the combined group significant decreases were found with regard to: triglycerides (TG) by 15%, total cholesterol (TC) by 25%, low-density lipoprotein cholesterol (LDL-C) by 41%, TC/high-density lipoprotein cholesterol (HDL-C) ratio by 29.5%, and increases in HDL-C by 7.5%. These changes were sustained until the end of study. After controlling for baseline levels, only the combined group, but not mono group, showed a significant difference (p<0.0001) in TC, LDL-C and TC/HDL-C ratio when compared with the placebo group. In summary, this study provides long-term efficacy of nattokinase supplementation and shows that the combined formula has relatively more potent effects than the mono formula on lowering of blood lipids, suggesting that combined nattokinase with RYR will be a better neutraceutical for patients with hyperlipidemia than nattokinase alone.

Nattokinase decreases plasma levels of fibrinogen, factor VII, and factor VIII in human subjects.

Nattokinase, a serine proteinase from Bacillus subtilis, is considered to be one of the most active functional ingredients found in natto. In this study, we hypothesized that nattokinase could reduce certain factors of blood clotting and lipids that are associated with an increase risk for cardiovascular disease (CVD). Thus, an open-label, self-controlled clinical trial was conducted on subjects of the following groups: healthy volunteers (Healthy Group), patients with cardiovascular risk factors (Cardiovascular Group), and patients undergoing dialysis (Dialysis Group). All subjects ingested 2 capsules of nattokinase (2000 fibrinolysis units per capsule) daily orally for 2 months. The laboratory measurements were performed on the screening visit and, subsequently, regularly after the initiation of the study. The intent-to-treat analysis was performed on all 45 enrolled subjects. By use of mixed model analysis, a significant time effect, but not group effect, was observed in the change from baseline of fibrinogen (P = .003), factor VII (P < .001), and factor VIII (P < .001), suggesting that the plasma levels of the 3 coagulation factors continuously declined during intake; also, the extents of decrease were similar between groups. After 2 months of administration, fibrinogen, factor VII, and factor VIII decreased 9%, 14%, and 17%, respectively, for the Healthy Group; 7%, 13%, and 19%, respectively, for the Cardiovascular Group; and 10%, 7%, and 19%, respectively, for the Dialysis Group, whereas blood lipids were unaffected by nattokinase. No significant changes of uric acid or notable adverse events were observed in any of the subjects. In summary, this study showed that oral administration of nattokinase could be considered as a CVD nutraceutical by decreasing plasma levels of fibrinogen, factor VII, and factor VIII.

Cerebellar hemorrhage provoked by combined use of nattokinase and aspirin in a patient with cerebral microbleeds.

Nattokinase is used as a health-promoting medicine for preventing thrombosis due to its fibrinolytic activity. Cerebral microbleed is remnant of blood extravasations from the damaged vessels related to cerebral microangiopathies. We report a patient, having used aspirin for secondary stroke prevention, who had an acute cerebellar hemorrhage after taking nattokinase 400 mg daily for 7 consecutive days. In addition to the hemorrhagic lesion, multiple microbleeds were demonstrated on brain MR images. We suggest that nattokinase may increase risk of intracerebral hemorrhage in patients who have bleeding-prone cerebral microangiopathy and are receiving other antithrombotic agent at the same time.

Prevention of venous thrombosis in long-haul flights with Flite Tabs: the LONFLIT-FLITE randomized, controlled trial.

The aim of this study was to evaluate the development of edema, and superficial and deep vein thrombosis (DVT) prophylaxis with an oral profibrinolytic agent (Flite Tabs, 150 mg pinokinase, Aidan, Tempe, AZ, USA) in long-haul flights (7-8 hours), in high-risk subjects. A group of 300 subjects was included; 76 were excluded for several problems including concomitant treatments; 204 were randomized into 2 groups (active treatment or placebo) to evaluate the effects of prophylaxis with Flite Tabs. An exercise program was used in both groups. The femoral, popliteal, tibial, and superficial veins were scanned with ultrasound before and within 90 minutes after flights. Of the included subjects, 92 of 103 controls and 94 of 101 treated subjects completed the study. Dropouts were due to connection problems. Age, gender, and risk distribution were comparable in the groups. In the treatment group, no DVT was observed. In the control group, 5 subjects (5.4%) had a DVT and there were 2 superficial thromboses (7 events in 92 subjects; 7.6%). At inclusion, edema was comparable in the 2 groups. After flights there was an increase in score in controls (+12%) in comparison with a decrease (-15%) in the Flite Tabs group (the difference in variation was statistically significant). Intention-to-treat analysis for thrombotic events shows 18 failures in controls (11 lost to follow-up + 7 thrombotic events) of 92 subjects (19.6%) in comparison with 7 failures (of 94 subjects, equivalent to 7.4%) in the treatment group (p < 0.05). Events were asymptomatic. In conclusion, Flite Tabs were effective in reducing thrombotic events and in controlling edema in high-risk subjects in long flights.

Proteolytic detergent enzymes enhance the allergic antibody responses of guinea pigs to nonproteolytic detergent enzymes in a mixture: implications for occupational exposure.

A guinea pig intratracheal test was developed to assess the respiratory allergenicity of enzymes used in the detergent industry. Information gained from this test was used in a process for setting operational exposure guidelines to protect worker health. Mixtures of enzyme proteins were given to guinea pigs once per week for 10 weeks to determine whether there were interactions among enzymes that affected the induction of antibody responses to each enzyme in the mixture. Passive cutaneous anaphylaxis antibody titers against each enzyme were measured in sera. Mixtures of two or three enzymes always consisted of a protease (Alcalase, Savinase; Novo Industri A/S) with an alpha-amylase (Termamyl; Novo Industri A/S), a lipase (Lipolase; Novo Industri A/S), or both. Control animals were exposed to single enzymes. The antibody titers to Termamyl and Lipolase were significantly greater in animals dosed with the protease-containing mixtures as compared with control animals dosed with a single enzyme. Antibody titers to the protease were unchanged in the presence of additional enzymes in the mixture. Complete inactivation of protease activity abrogated the enhanced antibody response to Lipolase. Inhalation exposure of guinea pigs to a mixture of Alcalase and Lipolase also resulted in higher antibody titers to Lipolase as compared with animals exposed by inhalation to Lipolase alone, showing that the enhanced response was not due to intratracheal delivery of antigen to the respiratory tract. These results show that proteolytic enzymes in a mixture enhance antibody responses to other enzymes. This should be considered when defining exposure guidelines for protease-containing enzyme mixtures.

Isolated late asthmatic reaction after exposure to a high-molecular-weight occupational agent, subtilisin.

High-molecular-weight agents generally induce immediate asthmatic reactions. We report the case of a subject who experienced a reaction that started after the first hour following exposure to subtilisin, a high-molecular-weight occupational agent. Any occurrence of immediate reaction was ruled out by measuring both FEV1 and lung volumes every 10 min in the first hour. This reaction was IgE-mediated as shown by immediate skin reactivity and increased specific IgE levels.

Exaggerated abrasion/erosion of human dental enamel surfaces: a case report.

An atypical, rapidly proceeding abrasion/erosion of the labial enamel surfaces of the maxillary and mandibular incisors and canines in a 27-yr-old man is reported. Ultrastructural examination of a replica of the teeth showed a practically structureless enamel surface both at the initial examination and after 12 months. However, at the end of the period, minor areas of dentin tubules became visible, indicating that a substantial loss of the tooth substance had taken place. The patient's occupation involved daily environmental contact with proteolytic enzymes. In vitro study of enamel exposed to one of the actual proteolytic enzymes showed dissolution of enamel substance, and it cannot be excluded that enzymatic decomposition of the organic enamel matrix is a contributing cause of the observed exaggerated loss of tooth substance.

Asthmagenic properties of a newly developed detergent ingredient: sodium iso-nonanoyl oxybenzene sulphonate.

The suspicion that a newly developed detergent ingredient, sodium iso-nonanoyl oxybenzene sulphonate (SINOS), was inducing asthma among a workforce led to a series of inhalation challenge tests to determine the specificity and dose response characteristics of its asthma provoking properties. Three previously exposed workers, three non-exposed non-asthmatic controls, and three non-exposed asthmatic controls were challenged with SINOS 0.01-100 micrograms and another chemically similar surface active detergent ingredient, linear alkyl benzene sulphonate (LAS) 0.01-100 micrograms. Asthmatic symptoms, late falls in FEV1, and increases in non-specific bronchial responsiveness were seen after the inhalation of SINOS in all three workers, confirming SINOS as a cause of occupational asthma. No changes were seen after the inhalation of SINOS in either group of control subjects nor after LAS in any subject. These findings suggest that SINOS causes asthma through a specific hypersensitivity mechanism unrelated to its surface active properties.

Possible adverse reactions to an enzyme-containing washing powder.

Following the launch of an enzyme (alcalase) containing washing powder, the manufacturers received numerous complaints from domestic users suggesting that it was responsible either for the precipitation of a rash or the exacerbation of a pre-existing rash. The 255 individuals living in London postal districts who complained to the manufacturers were contacted: 80 of them agreed to be investigated by patch and prick testing, and to a user test involving the double blind wearing of vests washed with the product. The results failed to substantiate that the enzyme containing washing powder was responsible for any dermatological problem.

A longitudinal study of possible allergy to enzyme detergents.

One hundred and thirty-six subjects with asthma and/or hay fever were surveyed for possible sensitization to the Bacillus licheniformis protease present in the commercial antigenically identical enzymes (Alcalase and Maxatase) used in household detergents. Prick-test reactions reported as minimally positive were given by one subject to both commercial enzyme preparations at the same standard non-irritating concentrations and by four others to one but not the other antigenically identical enzyme. Two of the five, and an additional seven, gave weak prick tests to the purified enzyme (Koch-Light Protease) at test protein concentrations 100 times stronger and to which non-specific irritation occurs. The findings are strongly against interpretation of these reactions as positive and were on no occasion comparable with those in sensitive workers. The skin-test reactions are attributable to non-specific effects. Neither enzyme-specific RAST IgE tests nor clinical histories suggested clinical sensitivity. Enzyme-specific RAST IgE for eighty-eight subjects over a period of 5 years showed no correlation with exposure. None had developed specific IgE levels like those of sensitized workers, and their RAST levels did not differ significantly from cord blood, unexposed subjects and prick-test-negative workers. The eight with the largest increase in specific IgE were prick-test-negative and their clinical reactions to enzyme detergents were comparable to those of non-enzyme detergents. There is no evidence from this study that exposure to modern, non-dusty household enzyme-containing detergents is likely to lead to clinical sensitization.

Scanning electron microscopic studies of the nasal mucosa in normal rabbit and rabbit exposed to alcalase.

The authors investigated the epithelium of the nasal mucous membrane with SEM to enable critical evaluation of alterations induced by the allergic reactions following endonasal application of Alcalase in rabbits. Experimental animals were previously submitted to 6-week intraperitoneal Alcalase sensitization, Biopsy specimens of the mucous membrane of the nasal septum were studied. Our study had confirmed alterations occurring in the epithelium following anaphylactic reaction to Alcalase. The significant morphological changes observed were formation of cytoplasm protuberances in the epithelial cells, and irregular epithelium with widened intercellular spaces, ladin leading to destruction of the respiratory epithelium.

Detergent enzymes and occupational safety. Observations on sensitization during Esperase production.

During a 10-year survey of 667 workers producing the detergent enzyme Esperase, derived from an alkolophilic strain of Bacillus licheniformis, 31 were found to have been sensitized. All but one were at the same time sensitized to subtilisins, with which they also had been working. No distinction could be made between symptoms attributed to Esperase and symptoms attributed to enzymes derived from Bacillus subtilis (licheniformis) or other enzymes. Symptoms, when present, were mainly from the lower respiratory tract. Nine sensitized workers were symptom-free. Sensitization was by IgE antibodies and detected by the RAST test. Twenty-one sensitized workers were transferred for precautionary measures or left the company. All 26 sensitized workers, whom it was possible to follow and reinvestigate, were no longer RAST-positive. Ten workers remained in their jobs. No signs of deteriorating lung function or permanent lung damage were found. The study indicated that, when strictly enforced, recommended operating procedures for workers handling hitherto well-known detergent enzymes are sufficient for dealing with Esperase. The data suggest there is no significant risk of consumer sensitization.