Fabrication of a Simple and Efficient HPLC Reduction Column for Online Conversion of Aristolochic Acids to Aristolactams Prior to Sensitive Fluorescence Detection.

Aristolochic acids (AAs) are nephrotoxic and carcinogenic nitrophenanthrene carboxylic acids produced naturally by plants from the Aristolochia and Asarum genera, which have been used extensively as herbal medicines. In addition to consuming AA-containing herbal medicinal products, there is emerging evidence that humans are also exposed to AA through the environment. In 2022, the World Health Organization (WHO) called for global action to remove AA exposure sources and to implement preventative measures against the development of AA-associated cancers. Herein, we report the development of a simple and efficient iron powder-packed reduction column that allows online post-column conversion of the nonfluorescing AA to its corresponding strongly fluorescing aristolactam (AL), facilitating the sensitive and selective detection of AA in herbal medicinal products, food grain, arable soil, or groundwater samples by high-performance liquid chromatography with fluorescence detection. Moreover, AL, a group of naturally occurring derivatives of AA that have demonstrated toxicity to cultured bacteria, human cells, and rats, is monitored and quantified simultaneously with AA in one single run without sacrificing sensitivity. In comparison with existing analytical methods for AA measurement, the newly developed method is not only inexpensive and less laborious, but it also offers improved sensitivity. We believe this novel method will find wide application in identifying the presence of AA in food, herbal medicines, and environmental samples, thus assisting in the identification and removal of AA exposure sources.

Effects of Diet on Aristolochic Acid-DNA Adduct Formation: Implications for Balkan Endemic Nephropathy Etiology.

Prolonged exposure to aristolochic acids (AAs) through AA-containing herbal medicine or AA-contaminated food is associated with the development of aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), both public health risks to which the World Health Organization is calling for global action to remove exposure sources. The AA exposure-induced DNA damage is believed to be related to both the nephrotoxicity and carcinogenicity of AA observed in patients suffering from BEN. While the chemical toxicology of AA is well-studied, we investigated in this study the understated effect of different nutrients, food additives, or health supplements on DNA adduct formation by aristolochic acid I (AA-I). By culturing human embryonic kidney cells in an AAI-containing medium enriched with different nutrients, results showed that cells cultured in fatty acid-, acetic acid-, and amino acid-enriched media produced ALI-dA adducts at significantly higher frequencies than that cultured in the normal medium. ALI-dA adduct formation was most sensitive to amino acids, indicating that amino acid- or protein-rich diets might lead to a higher risk of mutation and even cancer. On the other hand, cells cultured in media supplemented with sodium bicarbonate, GSH, and NAC reduced ALI-dA adduct formation rates, which sheds light on their potential use as risk-mitigating strategies for people at risk of AA exposure. It is anticipated that the results of this study will help to better understand the effect of dietary habits on cancer and BEN development.

Aristolochic acid I as an emerging biogenic contaminant involved in chronic kidney diseases: A comprehensive review on exposure pathways, environmental health issues and future challenges.

Described in the 1950s, Balkan Endemic Nephropathy (BEN) has been recognized as a chronic kidney disease (CKD) with clinical peculiarities and multiple etiological factors. Environmental contaminants - aromatic compounds, mycotoxins and phytotoxins like aristolochic acids (AAs) - polluting food and drinking water sources, were incriminated in BEN, due to their nephrotoxic and carcinogenic properties. The implication of AAs in BEN etiology is currently a highly debated topic due to the fact that they are found within the Aristolochiaceae plants family, used around the globe as traditional medicine and they were also incriminated in Aristolochic Acid Nephropathy (AAN). Exposure pathways have been investigated, but it is unclear to what extent AAs are acting alone or in synergy with other cofactors (environmental, genetics) in triggering kidney damage. Experimental studies strengthen the hypothesis that AAI, the most studied compound in the AAs class, is a significant environmental contaminant and a most important causative factor of BEN. The aim of this review is to compile information about the natural exposure pathways to AAI, via traditional medicinal plants, soil, crop plants, water, food, air. Data that either supports or contradicts the AAI theory concerning BEN etiology was consolidated and available solutions to reduce human exposure were discussed. Because AAI is a phytotoxin with physicochemical properties that allow its transportation in environmental matrices from different types of areas (endemic, nonendemic), and induce CKDs (BEN, AAN) and urinary cancers through bioaccumulation, this review aims to shed a new light on this compound as a biogenic emerging pollutant.

Differences in activated clotting time and total unfractionated heparin dose during pulmonary vein isolation in patients on different anticoagulation therapy.

BACKGROUND: Periprocedural pulmonary vein isolation (PVI) anticoagulation requires balancing between bleeding and thromboembolic risk. Intraprocedural anticoagulation is monitored by activated clotting time (ACT) with target value >300 s, and there are no guidelines specifying an initial unfractionated heparin (UFH) dose. METHODS: We aimed to assess differences in ACT values and UFH dosage during PVI in patients on different oral anticoagulants. We conducted an international, multi-center, registry-based study. Consecutive patients with atrial fibrillation (AF) undergoing PVI, on uninterrupted anticoagulation therapy, were analyzed. Before transseptal puncture, UFH bolus of 100 IU/kg was administered regardless of the anticoagulation drug. RESULTS: Total of 873 patients were included (median age 61 years, IQR 53-66; female 30%). There were 248, 248, 189, 188 patients on warfarin, dabigatran, rivaroxaban, and apixaban, respectively. Mean initial ACT was 257 ± 50 s, mean overall ACT 295 ± 45 s and total UFH dose 158 ± 60 IU/kg. Patients who were receiving warfarin and dabigatran compared to patients receiving rivaroxaban and apixaban had: (i) significantly higher initial ACT values (262 ± 57 and 270 ± 48 vs. 248 ± 42 and 241 ± 44 s, p < .001), (ii) significantly higher ACT throughout PVI (309 ± 46 and 306 ± 44 vs. 282 ± 37 and 272 ± 42 s, p < .001), and (iii) needed lower UFH dose during PVI (140 ± 39 and 157 ± 71 vs. 171 ± 52 and 172 ± 70 IU/kg). CONCLUSION: There are significant differences in ACT values and UFH dose during PVI in patients receiving different anticoagulants. Patients on warfarin and dabigatran had higher initial and overall ACT values and needed lower UFH dose to achieve adequate anticoagulation during PVI than patients on rivaroxaban and apixaban.

Fluoroscopy usage in contemporary interventional electrophysiology: Insights from a European registry.

BACKGROUND: Fluoroscopy has been an essential part of every electrophysiological procedure since its inception. However, till now no clear standards regarding acceptable x-ray exposure nor recommendation how to achieve them have been proposed. HYPOTHESIS: Current norms and quality markers required for optimal clinical routine can be identified. METHODS: Centers participating in this Europe-wide multicenter, prospective registry were requested to provide characteristics of the center, operators, technical equipment as well as procedural settings of consecutive cases. RESULTS: Twenty-five centers (72% university clinics, with a mean volume of 526 ± 348 procedures yearly) from 14 European countries provided data on 1788 cases [9% diagnostic procedures (DP), 38% atrial fibrillation (AF) ablations, 44% other supraventricular (SVT) ablations, and 9% ventricular ablations (VT)] conducted by 95 operators (89% male, 41 ± 7 years old). Mean dose area product (DAP) and time was 304 ± 608 cGy*cm2 , 3.6 ± 4.8 minutes, 1937 ± 608 cGy*cm2 , 15.3 ± 15.5 minutes, 805 ± 1442 cGy*cm2 , 10.6 ± 10.7 minutes, and 1277 ± 1931 cGy*cm2 , 10.4 ± 12.3 minutes for DP, AF, SVT, and VT ablations, respectively. Seven percent of all procedures were conducted without any use of fluoroscopy. Procedures in the lower quartile of DAP were performed more frequently by female operators (OR 1.707, 95%CI 1.257-2.318, P = .001), in higher-volume center (OR 1.001 per one additional procedure, 95%CI 1.000-1.001, P = .002), with the use of 3D-mapping system (OR 2.622, 95%CI 2.053-3.347, P < .001) and monoplane x-ray system (OR 2.945, 95%CI 2.149-4.037, P < .001). CONCLUSION: Exposure to ionizing radiation varies widely in daily practice for all procedure. Significant opportunities for harmonization of exposure toward the lower range has been identified.

Balkan Endemic Nephropathy and the Causative Role of Aristolochic Acid.

Balkan endemic nephropathy is a chronic tubulointerstitial disease with insidious onset, slowly progressing to end-stage renal disease and frequently associated with urothelial carcinoma of the upper urinary tract (UTUC). It was described in South-East Europe at the Balkan peninsula in rural areas around tributaries of the Danube River. After decades of intensive investigation, the causative factor was identified as the environmental phytotoxin aristolochic acid (AA) contained in Aristolochia clematitis, a common plant growing in wheat fields that was ingested through home-baked bread. AA initially was involved in the outbreak of cases of rapidly progressive renal fibrosis reported in Belgium after intake of root extracts of Aristolochia fangchi imported from China. A high prevalence of UTUC was found in these patients. The common molecular link between Balkan and Belgian nephropathy cases was the detection of aristolactam-DNA adducts in renal tissue and UTUC. These adducts are not only biomarkers of prior exposure to AA, but they also trigger urothelial malignancy by inducing specific mutations (A:T to T:A transversion) in critical genes of carcinogenesis, including the tumor-suppressor TP53. Such mutational signatures are found in other cases worldwide, particularly in Taiwan, highlighting the general public health issue of AA exposure by traditional phytotherapies.

Aristolochic Acids: Newly Identified Exposure Pathways of this Class of Environmental and Food-Borne Contaminants and its Potential Link to Chronic Kidney Diseases.

Aristolochic acids (AAs) are nitrophenanthrene carboxylic acids naturally produced by Aristolochia plants. These plants were widely used to prepare herbal remedies until AAs were observed to be highly nephrotoxic and carcinogenic to humans. Although the use of AA-containing Aristolochia plants in herbal medicine is prohibited in countries worldwide, emerging evidence nevertheless has indicated that AAs are the causative agents of Balkan endemic nephropathy (BEN), an environmentally derived disease threatening numerous residents of rural farming villages along the Danube River in countries of the Balkan Peninsula. This perspective updates recent findings on the identification of AAs in food as a result of the root uptake of free AAs released from the decayed seeds of Aristolochia clematitis L., in combination with their presence and fate in the environment. The potential link between AAs and the high prevalence of chronic kidney diseases in China is also discussed.

Balkan endemic nephropathy and aristolochic acid I: an investigation into the role of soil and soil organic matter contamination, as a potential natural exposure pathway.

Aristolochic acids (AAs) are carcinogenic and nephrotoxic plant alkaloids present in Aristolochia species, used in traditional medicine. Recent biomolecular and environmental studies have incriminated these toxins as an etiological agent in Balkan endemic nephropathy (BEN), a severe kidney disease occurring in the Balkan Peninsula. The questions on how the susceptible populations are exposed to these toxins have not yet been clearly answered. Exposure to AAs through the food chain, and environmental pollution (soil/dust), could provide an explanation for the presence of BEN in the countries where no folkloric use of the plant has been documented (Bulgaria, Croatia). Additional exposure pathways are likely to occur, and we have shown previously that AAs can contaminate crop plants through absorption from soil, under controlled laboratory environment. Here, we attempt to provide additional support to this potential exposure pathway, by revealing the presence of AAI in soil and soil organic matter samples collected from BEN and non-BEN areas. The samples were processed in order to be analyzed by high-pressure liquid chromatography, and ion trap mass spectrometry. Our results showed the presence of AAI in small concentrations, both in BEN and non-BEN soils, especially where Aristolochia plants and seeds were present.

Fluoroscopy-Free Pulmonary Vein Isolation in Patients with Atrial Fibrillation and a Patent Foramen Ovale Using Solely an Electroanatomic Mapping System.

INTRODUCTION: The advent of electroanatomical mapping (EAM) systems for pulmonary vein isolation (PVI) has dramatically decreased radiation exposure. However, the need for some fluoroscopy remains for obtaining left atrial (LA) access. The aim was to test the feasibility of fluoroscopy-free PVI in patients with atrial fibrillation (AF) and a patent foramen ovale (PFO) guided solely by an EAM system. METHODS: Consecutive patients with AF undergoing PVI and documented PFO were studied. An EAM-guided approach without fluoroscopy and ultrasound was used. After completing the map of the right atrium, the superior vena cava and the coronary sinus, a catheter pull-down to the PFO was performed allowing LA access. The map of the LA and subsequent PVI was also performed without fluoroscopy. RESULTS: 30 patients [age 61±12 years, 73% male, ejection fraction 0.64 (0.53-0.65), LA size in parasternal long axis 38±7 mm] undergoing PVI were included. The time required for right atrial mapping including transseptal crossing was 9±4 minutes. Total procedure time was 127±37 minutes. Fluoroscopy-free PVI was feasible in 26/30 (87%) patients. In four patients, fluoroscopy was needed to access (n = 3) or to re-access (n = 1) the LA. In these four patients, total fluoroscopy time was 5±3 min and the DAP was 14.9±13.4 Gy*cm2. Single-procedure success rate was 80% (24/30) after a median follow-up of 12 months. CONCLUSION: In patients with a documented PFO, completely fluoroscopy-free PVI is feasible in the vast majority of cases.

Effective reduction of fluoroscopy duration by using an advanced electroanatomic-mapping system and a standardized procedural protocol for ablation of atrial fibrillation: 'the unleaded study'.

AIMS: It is recommended to keep exposure to ionizing radiation as low as reasonably achievable. The aim of this study was to determine whether fluoroscopy-free mapping and ablation using a standardized procedural protocol is feasible in patients undergoing pulmonary vein isolation (PVI). METHODS AND RESULTS: Sixty consecutive patients were analysed: Thirty consecutive patients undergoing PVI using Carto3 were treated using a standardized procedural fluoroscopy protocol with X-ray being disabled after transseptal puncture (Group 1) and compared with a set of previous 30 consecutive patients undergoing PVI without a specific recommendation regarding the use of fluoroscopy (Group 2). The main outcome measures were the feasibility of fluoroscopy-free mapping and ablation, total fluoroscopy time, total dose area product (DAP), and procedure time. Sixty patients (age 60 ± 10 years, 73% male, ejection fraction 0.55 ± 0.09, left atrium 42 ± 8 mm) were included. In Group 1, total fluoroscopy time was 4.2 (2.6-5.6) min and mapping and ablation during PVI without using fluoroscopy was feasible in 29 of 30 patients (97%). In Group 2, total fluoroscopy time was 9.3 (6.4-13.9) min (P < 0.001). Total DAP was 13.2 (6.2-22.2) Gy*cm(2) in Group 1 compared with 17.5 (11.7-29.7) Gy*cm(2) in Group 2 (P = 0.036). Total procedure time did not differ between Groups 1 (133 ± 37 min) and 2 (134 ± 37 min, P = 0.884). CONCLUSION: Performing mapping and ablation guided by an electroanatomic-mapping system during PVI without using fluoroscopy after transseptal puncture using a standardized procedural protocol is feasible in almost all patients and is associated with markedly decreased total fluoroscopy duration and DAP.

Possible health impacts of naturally occurring uptake of aristolochic acids by maize and cucumber roots: links to the etiology of endemic (Balkan) nephropathy.

Aristolochic acids (AAs) are nephrotoxic and carcinogenic derivatives found in several Aristolochia species. To date, the toxicity of AAs has been inferred only from the effects observed in patients suffering from a kidney disease called "aristolochic acid nephropathy" (AAN, formerly known as "Chinese herbs nephropathy"). More recently, the chronic poisoning with Aristolochia seeds has been considered to be the main cause of Balkan endemic nephropathy, another form of chronic renal failure resembling AAN. So far, it was assumed that AAs can enter the human food chain only through ethnobotanical use (intentional or accidental) of herbs containing self-produced AAs. We hypothesized that the roots of some crops growing in fields where Aristolochia species grew over several seasons may take up certain amounts of AAs from the soil, and thus become a secondary source of food poisoning. To verify this possibility, maize plant (Zea mays) and cucumber (Cucumis sativus) were used as a model to substantiate the possible significance of naturally occurring AAs' root uptake in food chain contamination. This study showed that the roots of maize plant and cucumber are capable of absorbing AAs from nutrient solution, consequently producing strong peaks on ultraviolet HPLC chromatograms of plant extracts. This uptake resulted in even higher concentrations of AAs in the roots compared to the nutrient solutions. To further validate the measurement of AA content in the root material, we also measured their concentrations in nutrient solutions before and after the plant treatment. Decreased concentrations of both AAI and AAII were found in nutrient solutions after plant growth. During this short-term experiment, there were much lower concentrations of AAs in the leaves than in the roots. The question is whether these plants are capable of transferring significant amounts of AAs from the roots into edible parts of the plant during prolonged experiments.

Electrophysiological predictors of propafenone efficacy in prevention of atrioventricular nodal re-entrant and atrioventricular re-entrant tachycardia.

AIM: To assess the efficacy of propafenone in prevention of atrioventricular nodal reentrant tachycardia (AVNRT) and orthodromic atrioventricular tachycardia (AVRT) based on the clinical results of arrhythmia recurrence and find the electrophysiological predictor of propafenone effectiveness. METHODS: This retrospective study included 44 participants in a 12-month period, who were divided in two groups: group A - in which propafenone caused complete ventriculo-atrial block and group B - in which propafenone did not cause complete ventriculo-atrial block. RESULTS: Group A had significantly lower incidence of tachycardia than group B (95% vs 70.8%, P=0.038), and complete ventriculo-atrial block predicted the efficacy of propafenone oral therapy in the prevention of tachycardia (sensitivity 87.5%, specificity 52.8%, positive predictive value 95%, negative predictive value 29.2%). Patients with AVNRT in group B who did not experience the recurrences of tachycardia had significantly shorter echo zone before intravenous administration of propafenone than the patients who experienced episodes of sustained tachycardia (median 40 ms [range 15-60 ms] vs 79 ms [range 50-180 ms], P=0.008). CONCLUSION: In patients with non-inducible tachycardia, complete ventriculo-atrial block can be used as an electrophysiological predictor of the efficacy of propafenone oral therapy in the prevention of tachycardia. In patients with non-inducible AVNRT, but without complete ventriculo-atrial block, propafenone was more effective in patients with shorter echo zone of tachycardia.

Electrophysiological effects, efficacy and safety of intravenous propafenone in termination of atrioventricular nodal reentrant tachycardia and atrioventricular reentrant tachycardia: a prospective non-randomized interventional study.

The aim of this prospective, non-randomized interventional study was to assess electrophysiological effects, efficacy and safety of intravenous propafenone in termination of atrioventricular nodal reentrant tachycardia (AVNRT) and orthodromic atrioventricular reentrant tachycardia (AVRT). This single-center study was carried out at Department of Cardiology, Sestre milosrdnice University Hospital in Zagreb, Croatia, between January 1, 2005 and December 31, 2006. Eligibility requirements were fulfilled by a total of 70 patients with AVNRT (n=37) and AVRT (n=33). The intervention consisted of the electrophysiological study aimed at inducing tachycardia, followed by intravenous administration of 2 mg/kg propafenone in both groups. The main outcome measures were safety and efficacy of 2 mg/kg intravenous propafenone in tachycardia termination and re-induction. Out of 37 patients with AVNRT, propafenone managed to terminate it in 28 (75.7%) patients, while tachycardia was not inducible in 25 (67.56%) patients. Out of 33 patients with AVRT, propafenone managed to terminate AVRT in 29 (87.9%) patients, while tachycardia was not inducible in 22 (66.66%) patients. The overall propafenone efficacy in tachycardia termination was 81.42%. No propafenone-related adverse effects were recorded during the study period. Propafenone was found to be a safe and effective anti-arrhythmic drug and can be justifiably administered for AVNRT and AVRT termination. It could be considered as an alternative to adenosine and verapamil.